Compositions and methods for regulating glucose homeostasis and insulin action

ABSTRACT

The present invention encompasses the use of compounds for a novel approach to treat and prevent diseases, conditions, and disorders such as diabetes and ischemic reperfusion injury. Compounds of the invention, including but not limited to BAM15 ((2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine), a mitochondrial uncoupler, can improve glucose tolerance, increases cellular oxygen consumption, treat or prevent kidney ischemia reperfusion injury reverse insulin resistance, reverse or treat hyperinsulinemia, and reverse or treat hyperlipidemia. The present invention further provides novel compounds as well as methods for identifying compounds with the same or similar properties as BAM15.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 16/387,268,filed Apr. 17, 2019; which is a Continuation of U.S. application Ser.No. 15/341,961, filed Nov. 2, 2016; which is a Continuation ofapplication Ser. No. 14/409,793, filed Dec. 19, 2014; now U.S. Pat. No.9,492,448, which is a national stage filing of International ApplicationNo. PCT/2013/046740, filed Jun. 20, 2013, which claims the benefit ofU.S. Provisional Application No. 61/662,268, filed Jun. 20, 2012 and allthe benefits accruing therefrom under 35 U.S.C. § 119(e), the content ofwhich is incorporated by reference in its entirety.

BACKGROUND

Type 2 diabetes (T2D) is a chronic and progressive metabolic disordercharacterized by hyperglycemia and hyperinsulinemia. Obesity and reducedphysical activity are major contributors to insulin resistance,diabetes, and diabetes-related complications such as heart disease andrenal failure. It is estimated that over 300 million people worldwideand more than 8% of Americans are overweight and insulin resistantpre-diabetics. Although exercise and calorie restriction are veryeffective reversers of insulin resistance and T2D, these interventionshave poor patient compliance. Current anti-diabetes drugs fit into manyclasses of agents that increase insulin sensitivity, increase insulinsecretion, or reduce nutrient intake/absorption. These drugs improve T2Dsymptoms and extend patient lifespan; however, most diabetic patientseventually succumb to the complications of their disease. Recentsetbacks in diabetes therapy include the cardiovascular concerns withthe anti-diabetes drug rosiglitazone (Avandia) and the minimal advancesreported in several recent ‘mega’ clinical trials (e.g., ACCORD,NICE-SUGAR, ADVANCE, and VADT). As such, new pharmacologicalintervention in diabetes is needed.

In the 1930's, the ‘mitochondrial protonophore uncoupler’2,4-dinitrophenol (DNP) was widely prescribed as an anti-obesitytreatment to tens of thousands of people. DNP mimicked the beneficialeffects of diet and exercise by depleting intracellular nutrient stores,and, in so doing, it also had beneficial effects on glucose metabolism.Patients consuming˜300 mg/d steadily shed an average of 1.5 pounds perweek over the course of several months without changes in food intake.Similarly, mice treated with DNP demonstrate improved serologicalglucose, triglyceride, and insulin levels, as well as decreasedoxidative damage, reduced body weight, and increased longevity. Themechanism of mitochondrial uncoupling is inherently an antioxidantmechanism and consequently mitochondrial uncouplers such as DNP haveprotective effects on ischemia-reperfusion injury and other disordersrelated to mitochondrial reactive oxygen species production.Unfortunately, DNP has off-target effects on other cellular membranesresulting in a narrow therapeutic index. DNP was subsequently withdrawnfrom the North American market by the US Food and Drug Administration in1938. Currently, there are no uncoupler drugs that are safe enough foruse in humans.

Mitochondrial protonophore uncouplers are small molecules that transferprotons across the mitochondrial inner membrane (MIM). These moleculesare referred to as ‘uncouplers’ because they allow protons to re-enterthe mitochondrial matrix via a pathway independent of ATP synthase and,therefore, uncouple nutrient oxidation from ATP production.Pharmacologic uncouplers, when used at optimal concentrations, improvethe efficiency of the mitochondrial electron transport chain anddecrease mitochondrial reactive oxygen species (ROS) production. Themajor limitation of DNP and other protonophore uncouplers is theirunwanted protonophore activity at the plasma membrane (PM). Thisoff-target activity increases intracellular acidification, depolarizeselectrically stimulated cells, and increases energy demand needed tomaintain the cellular ion gradient. When these off-target effects arecombined with reduced efficiency of mitochondrial respiration the sideeffects include over-heating and ATP depletion. This clinical historywith DNP overdose has led to the misconception that all mitochondrialuncouplers will cause these side effects.

Mitochondria regulate cellular metabolism and play an important role inthe pathogenesis of some of the most prevalent human diseases includingobesity, cancer, diabetes, neurodegeneration, and heart disease. Many ofthese diseases can be improved by the use of pharmacological agents likemitochondrial proton transporters that lessen mitochondrial oxidativedamage and increase energy expenditure. Genetic and pharmacologicuncoupling have beneficial effects on disorders that are linked tomitochondrial oxidative stress, such as ischemic-reperfusion injury,Parkinson's disease, insulin resistance, aging, and heart failure, anddisorders that stand to benefit from increased energy expenditure suchas obesity. The development of a selective mitochondrial protonophoreuncoupler that does not affect the plasma membrane potential wouldbroaden the safety margin of mitochondrial uncouplers and providerenewed hope that mitochondrial uncoupling can be targeted for thetreatment of obesity, type II diabetes, and other diseases, disorders,and conditions related to mitochondrial function.

There is a long felt need in the art for compositions and methods usefulfor treating diabetes, regulating glucose homeostasis, reducingadiposity, protecting from ischemic-reperfusion injury, and regulatinginsulin action using mitochondrial uncouplers as well as for compoundsuseful as mitochondrial uncouplers. The present application satisfiesthese needs.

SUMMARY OF THE INVENTION

Disclosed herein is the discovery that BAM15 is a mitochondrialuncoupler. BAM15 demonstrates similar potency to the most potentuncoupler known, carbonyl cyanide p-trifluoromethoxyphenylhydrazone(FCCP), but does not have protonophore activity at the plasma membrane.As a result, BAM15 causes less intracellular acidification, lessmitochondrial toxicity, marked improvements in cell viability, and iseffective over a much wider concentration range than FCCP. Theseunprecedented properties have been long sought after and provide greatpotential for the treatment of mitochondria-related disorders,including, but not limited to, obesity, diabetes, insulin resistance,Parkinson's disease, aging, traumatic brain injury, ischemia-reperfusioninjury, and heart failure. The present invention discloses that someknown compounds, identified by library screening, have the unexpectedproperties of acting as mitochondrial uncouplers and further disclosesnovel methods to assay for these properties as well as provide novelcompounds that are analogs and derivatives of the compounds with theactivity disclosed herein.

In one embodiment, a compound of the invention is useful for treatingdisease, disorders, and conditions which are associated with defects inmitochondrial function or which can be treated with drugs or agents thatact as uncoupling agents.

In one embodiment, a compound of the invention can stimulate oxygenconsumption rate (OCR) when ATP synthase is inhibited. In oneembodiment, a compound of the invention can depolarize the mitochondrialinner membrane. In one embodiment, a compound of the invention canstimulate respiration in isolated mitochondria. In one embodiment, acompound of the invention can increase OCR without donating electrons tothe electron transport chain. In one aspect, the compound is BAM15 andanalogs and derivatives thereof. As disclosed herein, BAM15 stimulatesmitochondrial respiration in the presence of the ATP synthase inhibitoroligomycin in L6 rat myoblasts and NMuLi normal murine liver cells,BAM15 treatment of L6 myoblasts depolarized mitochondria, asdemonstrated by a leftward shift in fluorescence of the cationicmitochondrial membrane potential dye TMRM, BAM15 stimulated respirationin isolated mouse liver mitochondria respiring on either pyruvate andmalate, or succinate in the presence of the complex I inhibitorrotenone, and BAM15 was not an electron donor to the electron transportchain as determined by performing an ‘electron flow’ assay in thepresence of 5 μM of either BAM15 or FCCP (positive control). In oneaspect, a compound of the invention is an energy expenditure agonist. Inone aspect, a compound of the invention is an antioxidant.

It is disclosed herein that BAM15 has superior properties over otherprotonophore mitochondrial uncouplers because it acts at mitochondriaand lacks protonophore activity at the plasma membrane. Themitochondrial inner membrane selectivity of these potential drugs isimportant because uncoupling reduces the proton motive force andincreases the flow of electrons through the electron transport chain inthe mitochondria to accelerate respiration and maintain membranepotential. BAM15 is unrelated to known uncouplers and it outperforms themost potent uncoupler known, FCCP, in the contexts of improved cellviability and therapeutic range. Additionally, it is disclosed hereinthat BAM15 is devoid of plasma membrane protonophore activity.

Many anti-diabetes drugs such as insulin-sensitizers promote glucoseclearance from the blood by effectively ‘pushing’ glucose into nutrientoverloaded tissues; however, in contrast to this approach our strategyis aimed at reducing cellular nutrient stores so that tissues will‘pull’ glucose from the circulation. The present method is modeled afterexercise and calorie restriction interventions which also reducecellular nutrient stores to improve glycemia and insulin sensitivity.The proof of principle is validated in humans treated with themitochondrial uncoupler 2,4-dinitrophenol (DNP). DNP decreases adiposityand improves metabolism in humans; however, it also has a very narrowtherapeutic window and was removed from FDA approval in 1938. Otheranti-diabetes drugs including agonists of thyroid hormone and inhibitorsof 1143 hydroxysteroid dehydrogenase type 1 have off-target effects ofincreased energy expenditure that may mediate some of the protectiveeffects of these compounds. Nevertheless, there are no drugs have beenspecifically targeted for increased energy expenditure.

The compound BAM15 shows promising insulin sensitizing, anti-adiposity,and antioxidant effects in cultured cells and mice. Other compounds havebeen tested as well using the methods of the invention and new analogsand derivatives of BAM15 are disclosed herein.

Useful compounds of the invention include, but are not limited to,BAM15, BAM8, BAM9, BAM15A, BAM15B, BAM15C, BAM15D, BAM15E, BAM15F, FCCP,and 2,4-dinitrophenol, as well as biologically active analogs andderivatives thereof. Some of these compounds are new analogs of BAM15and are disclosed herein.

A compound of the invention has at least one of the following propertiesor activities: energy expenditure agonist, mitochondrial uncoupler,antioxidant, increases oxygen consumption, depolarizes the mitochondrialinner membrane, stimulates respiration in isolated mitochondria,increases or stimulates oxygen consumption without donating electrons tothe electron transport chain, lacks protonophore activity at the plasmamembrane, reduces reperfusion-induced mitochondrial oxidative stress andmitochondrial fragmentation, reduces cellular reactive oxygen species,improves glucose tolerance, provides protection from high fat induceglucose tolerance, activates AMPK without depletion of ATP, prevents,reverses or treats insulin resistance, prevents, reverses or treatshyperinsulinemia, prevents, reverses or treats hyperlipidemia, improvesblood lipid profiles, improves leanness, improves insulin sensitivity,protects from ischemic-reperfusion injury, and is less toxic than othermitochondrial inhibitors.

In one embodiment, a compound of the invention has the general formula:

as well as active analogs and derivatives thereof.

In one aspect, R₁-R₁₀ are all independently optional. In one aspect,each of R₁-R₁₀ is independently selected from the group consisting of:H, halogen, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, andamino acid (each group can be optionally substituted), or apharmaceutically acceptable salt or prodrug thereof. In one aspect, thehalogen is independently F, Cl, Br, or I. In one aspect it is F.

In one embodiment, a compound of the invention has the general formulaII:

as well as active analogs and derivatives thereof. In one aspect, R₁-R₂are independently optional. In one aspect, each is independentlyselected from the group consisting of: H, halogen, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, and amino acid (each group can beoptionally substituted), or a pharmaceutically acceptable salt orprodrug thereof. In one aspect, the halogen is independently F, Cl, Br,or I. In one aspect it is F.

One of ordinary skill in the art will appreciate that not allconfigurations need to be effective or as effective as other compoundsof the genus.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine compound activity using thestandard tests described herein, or using other similar tests which arewell known in the art.

In one embodiment, the present invention provides compositions andmethods for preventing or treating a disease, disorder, or condition,comprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, optionallyat least one additional therapeutic agent, and an effective amount of atleast one compound having a structure of Formula I or Formula II. In oneaspect, the disease, disorder or condition is selected from the groupconsisting of ischemia reperfusion injury, hyperinsulinemia,hyperlipidemia, glycemia, glucose tolerance, insulin sensitivity,adiposity, insulin resistance, obesity, diabetes, cancer,neurodegeneration, heart disease, renal disease, heart failure,Parkinson's disease, traumatic brain injury, stroke, aging, anddisorders standing to benefit from increased energy expenditure. In oneaspect, the compound is a mitochondrial uncoupler. In one aspect, thediabetes is type II diabetes. In one aspect, the ischemia reperfusioninjury is kidney ischemia reperfusion injury, cardiac ischemiareperfusion injury, or brain ischemia reperfusion injury. In one aspect,the brain ischemia reperfusion injury is related to stroke or traumaticbrain injury. In one aspect, the method reduces reperfusion-inducedmitochondrial oxidative stress and mitochondrial fragmentation.

Compounds of the invention can be administered to a subject at varioustimes, dosages, and more than once, depending on, for example, the age,sex, health, and weight of the subject, as well as on the particulardisease, disorder, or condition to be treated or prevented. In oneaspect, a compound is administered at a dosage ranging from about 0.1mg/kg to about 50 mg/kg body weight. In another aspect, the compound isadministered at a dosage ranging from about 0.5 mg/kg to about 25 mg/kgbody weight. In yet another aspect, the compound is administered at adosage ranging from about 1.0 mg/kg to about 5.0 mg/kg body weight. Inone aspect, about 3.0 mg/kg is administered. In another aspect, about5.0 mg/kg is administered. In another aspect, the compound isadministered as a unit dose ranging from about 10 mg to about 500mg/unit dose.

In one aspect, a compound is administered more than once. In one aspect,the compound is a mitochondrial protonophore uncoupler lackingprotonophore activity at the plasma membrane.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Processes for preparing compounds of a generic formula of the invention,such as formulas I or II, or for preparing intermediates useful forpreparing compounds of formula I or other formulas of the invention areprovided as further embodiments of the invention or are known in theart. Intermediates useful for preparing compounds of formula I or otherformulas are also provided as further embodiments of the invention.

Useful compounds of the invention include, but are not limited to:

BAM15 is further described at the national library of medicine websitein the “pubchem” section, where it is referred to as compound ID 565708.Properties of BAM15 include: Molecular Weight: 340.287006 [g/mol] andMolecular Formula: C₁₆H₁₀F₂N₆O. Its chemical names are(2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amineandN5,N6-bis(2-fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine(its IUPAC name).

Other useful compounds for aspects of the invention include FCCP and2,4-dinitrophenol, having the following structures:

In one embodiment, the present invention provides compositions andmethods for preventing or treating a disease, disorder, or condition,comprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, optionallyat least one additional therapeutic agent, and an effective amount of atleast one compound having a structure of Formula I or Formula II.

In one aspect, the compound is a mitochondrial uncoupler.

In one aspect, the disease, disorder or condition is selected from thegroup consisting of ischemia reperfusion injury, hyperinsulinemia,hyperlipidemia, glycemia, glucose tolerance, insulin sensitivity,adiposity, insulin resistance, obesity, diabetes, cancer,neurodegeneration, heart disease, renal disease, heart failure,Parkinson's disease, traumatic brain injury, stroke, aging, anddisorders standing to benefit from increased energy expenditure. In oneaspect, the compound is a mitochondrial uncoupler. In one aspect, thediabetes is type II diabetes. In one aspect, the ischemia reperfusioninjury is kidney ischemia reperfusion injury, cardiac ischemiareperfusion injury, or brain ischemia reperfusion injury. In one aspect,the brain ischemia reperfusion injury is related to stroke or traumaticbrain injury. In one aspect, the method reduces reperfusion-inducedmitochondrial oxidative stress and mitochondrial fragmentation.

Additional therapeutic agents include, but are not limited to, drugsused to treat infections or other drugs and agents used to treat orprevent the specific disease, disorder, or condition.

In one aspect, the diabetes is type II diabetes.

In one aspect, the ischemia reperfusion injury is kidney ischemiareperfusion injury, brain ischemia reperfusion injury, or cardiacischemia reperfusion injury.

In one aspect, the method reduces reperfusion-induced mitochondrialoxidative stress and mitochondrial fragmentation.

In one embodiment, a compound of the invention is selected from thegroup consisting of BAM15, BAM8, BAM9, BAM15A, BAM15B, BAM15C, BAM15D,BAM15E, BAM15F, FCCP, and 2,4-dinitrophenol, as well as biologicallyactive analogs and derivatives thereof:

In one embodiment, a compound of the invention is administered at adosage ranging from about 0.1 mg/kg to about 50 mg/kg body weight. Inone aspect, the compound is administered at a dosage ranging from about0.5 mg/kg to about 25 mg/kg body weight. In another aspect, the compoundis administered at a dosage ranging from about 1.0 mg/kg to about 5.0mg/kg body weight. In a further aspect, the compound is administered asa unit dose ranging from about 10 mg to about 500 mg. In one aspect, thecompound is administered more than once. A compound is administered byany suitable method.

In one embodiment, the compound is a mitochondrial protonophoreuncoupler lacking protonophore activity at the plasma membrane.

In one embodiment, the compound is BAM15:

In one embodiment, the compound increases oxygen consumption.

In one embodiment, the compound reduces cellular reactive oxygenspecies.

In one embodiment, the compound depolarizes the mitochondrial innermembrane.

In one embodiment, the compound increases oxygen consumption ratewithout directly donating electrons to the electron transport chain.

In one aspect, a pharmaceutical composition comprising an effectiveamount of at least one compound of the invention is administered to thesubject.

In one aspect, administration of a compound of the invention improvesglucose tolerance. In one aspect, BAM15 improves glucose tolerance. Inone aspect, administration of a compound of the invention providesprotection from high fat diet-induced glucose intolerance. In oneaspect, a compound of the invention is an agonist of energy expenditureand increases oxygen consumption without ROS production and activatesAMPK without depletion of ATP.

In one aspect, a compound of the invention increases cellular oxygenconsumption.

In one embodiment, a compound of the invention reverses insulinresistance. In one aspect, the compound reverses or treatshyperinsulinemia. In one aspect, the compound reverses or treatshyperlipidemia. In one aspect, a compound of the invention improvesglucose tolerance. In one aspect, a compound of the invention improvesglucose tolerance in a subject on a high fat diet. In one embodiment, acompound of the invention is useful for increasing cellular oxygenconsumption. In one embodiment, a compound of the invention is useful asan anti-diabetic.

In one aspect, administration of a compound of the invention to asubject in need thereof will cause improvements in blood lipid profiles,glucose tolerance, leanness, and insulin sensitivity. In one aspect,improvements in blood lipid profiles, glucose tolerance, leanness, andinsulin sensitivity occur without hypophagia. In one aspect,improvements in blood lipid profiles, glucose tolerance, leanness, andinsulin sensitivity occur without hyperinsulinemia. In one aspect,improvements in blood lipid profiles, glucose tolerance, leanness, andinsulin sensitivity occur without hypophagia or hyperinsulinemia. Theinvention therefore encompasses the use of BAM15 and other compounds ofthe invention having similar activity for use in treating and preventingobesity.

In one embodiment, a compound of the invention can be useful fortreating mitochondrial dysfunction.

In one embodiment, a compound of the invention is useful for decreasingreactive oxygen species production and in turn lessens ischemiareperfusion-mediated injury in a tissue.

A compound of the invention, such as BAM15, has certain properties thatcan be tested for and identified in other compounds using the methods ofthe invention. For example, a new compound of the invention will havethe measurable properties required of a mitochondrial protonophoreuncoupler when subjected to a series of biochemical assays such as theability to stimulate OCR when ATP synthase is inhibited, depolarize themitochondrial inner membrane, stimulate respiration in isolatedmitochondria, and increase OCR without donating electrons to theelectron transport chain.

In one aspect, a compound of the invention comprises a molecular weightbetween 205-370, HBA<5, HBD<3, 1-3 rings, and a calculated Log Sof >10-3. The present invention further provides compositions andmethods for identifying compounds comprising the activity describedherein. The novel screening assay is modeled upon the mechanisms ofaction of diet and exercise, including cellular nutrient composition,amplified antioxidant defense, and insulin sensitivity. In one aspect,the assay is exemplified by Example 1, FIG. 1. In another aspect, it isexemplified by the assay exemplified by Example 2, FIG. 5. For example,Example 2, FIG. 5 schematically illustrates a method to identify newmitochondrial uncoupler molecules with a broad range of cellular oxygenby first performing a dose response for oxygen consumption rate incells. Then, in order to validate protonophore activity and lack ofelectron donor activity in mitochondria, a complex coupling assay inisolated mitochondria is performed. At that point assays are performedto demonstrate that the test compound does not act at the plasmamembrane, that it does not cause cellular acidification, it does notcause reactive oxygen species production, and that it is not cytotoxicat effective doses, using assays including patch clamp, viabilityassays, pH assay, and ROS assays.

The present application further provides compositions comprising atleast one compound useful as a mitochondrial uncoupler, said compoundselected from the group consisting of compounds having a structure offormula I or formula II. In one embodiment, the compound is selectedfrom the group consisting of BAM15, BAM8, BAM9, BAM15A, BAM15B, BAM15C,BAM15D, BAM15E, BAM15F, FCCP, and 2,4-dinitrophenol, as well asbiologically active analogs and derivatives thereof. In one aspect, thecompound is BAM15. In one aspect, the compound lacks protonophoreactivity at the plasma membrane.

The present invention further provides compositions and methods foridentifying compounds with the properties required herein as well as fordetermining if a compound is a mitochondrial uncoupler with lowtoxicity. Steps include contacting a cell with a test mitochondrialuncoupler and measuring energy expenditure and when an increase inenergy expenditure is detected the test mitochondrial uncoupler issubjected to an assay to measure reactive oxygen species. Then, when thetest mitochondrial uncoupler does not increase energy expenditure viaproduction of reactive oxygen species, the test mitochondrial uncoupleris tested to determine effective dosing index and then the testmitochondrial uncoupler is tested for insulin sensitizing effects bymeasuring the ability to reverse a model of insulin resistance. Apositive result for reversing a model of insulin resistance is anindication that the compound is a mitochondrial uncoupler with lowtoxicity. In one aspect, increased energy expenditure is measured usingan oxygen consumption assay. In one aspect, the oxygen consumption assayused is performed in the presence of an O₂-sensitive fluorophore. In oneaspect, the oxygen consumption is measured using an extracellular fluxanalyzer. In one aspect, the compound increases oxygen consumption inthe presence of an ATP synthase inhibitor. In one aspect, the compoundlacks protonophore activity at the plasma membrane. The applicationfurther includes compounds identified by this method.

The present invention further provides a method of increasing oxygenconsumption, reducing cellular reactive oxygen species, depolarizing amitochondrial inner membrane, and increasing oxygen consumption ratewithout donating electrons to the electron transport chain using amitochondrial uncoupler, comprising contacting a cell or mitochondriawith a composition comprising at least one compound having a structureof formula I or formula II as disclosed herein. In one aspect, themethod increases extracellular acidification. In one aspect, thecompounds is selected from the group consisting of BAM15, BAM8, BAM9,BAM15A, BAM15B, BAM15C, BAM15D, BAM15E, BAM15F, FCCP, and2,4-dinitrophenol, as well as biologically active analogs andderivatives thereof. In one aspect, the compound lacks protonophoreactivity at the cell membrane. In one aspect, the compound is BAM15.

The present invention further provides kits for using the compounds andassays of the invention. In one embodiment, the invention provides a kitfor measuring mitochondrial respiration and glycolysis. The kit caninclude BAM15, optionally FCCP or other compounds useful in the assay ascontrol compounds, other reagents useful for measuring mitochondrial andglycolysis, optionally an extracellular flux analyzer, and aninstructional material describing the use of the kit. The kit can beused with an extracellular flux analyzer and appropriate programs, etc.In one embodiment, the kit provides for measuring mitochondrialrespiration. In one aspect, measuring mitochondrial respirationcomprises measuring basal respiration, ATP turnover, proton leak,maximal respiration, and spare respiratory capacity. In one aspect, thisincludes measuring one or more of the following-glycolysis, glycolyticcapacity, and glycolytic reserve. In one aspect, an extracellular fluxanalyzer is used or can be provided with the kit.

The present invention further encompasses compounds identified by themethods of the invention.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS Example 1

FIG. 1. Flow chart. Increased oxygen consumption is a major indicator ofenergy expenditure. The library was screened using BD biosciences oxygenbiosensor plates and confirmed the hits using a Seahorse XF24extracellular flux analyzer to assess cellular oxygen consumption. Hitswere then screened for reactive oxygen species (ROS) production. Thissecondary screen serves 2 purposes; to rule out compounds which increaseoxygen consumption via ROS production and to identify compounds that areantioxidants. Finally, insulin-sensitizing effects of hit compounds aredetermined by testing their ability to reverse multiple models ofinsulin resistance including hyperinsulinemia and hyperlipidemia.

FIG. 2. Hit compounds increase cellular oxygen consumption. Hitcompounds displayed a broad range of phenotypes. L6 myoblasts weretreated with a dose response of each hit and FCCP, a mitochondrialuncoupler, was used as a positive control. Two of our compounds (barn 8and barn 15) were more potent than fccp (red line). One characteristicof mitochondrial uncouplers is that they exhibit an inverted bell shapedcurve of effectiveness-they increase oxygen consumption up to a dosagethat inhibits mitochondrial function. This is observed with compoundsfccp and barn 8. The other compounds did not exhibit themitochondria-toxicity characteristic of pharmacologic uncouplers andseveral molecules have a therapeutic window over several orders ofmagnitude. Data shown are averages of 3 wells.

FIG. 3. Hit compound secondary screen for ROS production. L6 cells wereloaded with 10 μM CM-DCFDA, a ROS-sensitive dye, for 1 hour before beingwashed and treated with 10 uM of each compound for 1 hour. DMSO and 100nM hydrogen peroxide were used as negative and positive controls. Datashown are an average of three experiments.

FIG. 4. AMPK activation and cellular ATP levels after treatment with hitcompounds. L6 cells were treated with each hit compound (10 uM) andphosphorylation of AMPK was measured by Western blot (top panel).Oligomycin, an ATP synthase inhibitor, was used as a positive control.BAM7 was not available for purchase by the supplier. BAM16 thru BAM-25were not tested. 14-3-3 was blotted as a housekeeping loading control.Cellular ATP levels were measured after the seahorse XF24 oxygenconsumption experiment shown in FIG. 2 (> one hour with 10 uM-hitcompound).

Example 2

FIG. 5. (also referred to as Example 2, FIG. 1) BAM15 acts on isolatedmitochondria and is not an electron donor to the ETC. This ‘complexcoupling’ experiment starts with isolated mitochondria respiring onpyruvate and malate in the presence of FCCP or BAM15 (5 μM) at time 0.After 10 mins, 2 μM rotenone is added to inhibit ETC complex I. Rotenonedecreases oxygen consumption in mitochondria treated with either FCCP orBAM15 indicating that neither FCCP nor BAM15 donate electrons to the ETCdownstream of complex I. Succinate was then added at 20 min to stimulaterespiration from complex II. Neither FCCP nor BAM15 affect the increasein respiration indicating that they do not affect complex II. At 25 min,the mitochondria were treated with 4 μM antimycin A (AntA) to inhibitcomplex III and block succinate-mediated respiration. These datademonstrate that neither compound donates electrons from succinate tocytochrome c or complex IV. Finally, at 31 min the electron donor systemof ascorbate/TMPD was added to feed electrons to complex IV. In sum,these data indicate that BAM15 increases respiration in isolatedmitochondria via a mechanism that does not involve electron donation tothe ETC.

FIG. 6. (also referred to as Example 2, FIG. 2) BAM15 causes lessintracellular acidification than FCCP. Intracellular pH change following5 min of treatment with FCCP or BAM15 at indicated dosages (n=3 for allfigures). FIGS. 7A.-7D. (also referred to as Example 2, FIG. 3) BAM15 isless cytotoxic than FCCP. BAM15 and FCCP were administered to culturedcardiomyocytes, L6 cells, and NMuLi cells for 48 h at the indicatedconcentrations. In all cell lines viability was markedly improved inBAM15 cells (cardiomyocytes shown in FIG. 7A and L6 cells and NMuLicells shown in FIG. 7B, n=3). (FIG. 7C) BAM15 or FCCP were administeredto L6 cells at the indicated concentrations (in μM) for 40 min prior toanalysis of AMP-activated protein kinase phosphorylation (n=2). (FIG.7D) Cellular ATP levels were measured 20 min following FCCP or BAM15treatment (20 μM, n=2).

FIG. 8. (also referred to as Example 2, FIG. 4) BAM15 treatment improvesglucose tolerance and leanness in mice fed a high fat diet. (Left) At 8weeks of age, C57BL/6 mice were fed a high fat diet for 6 weeks prior todaily treatment with BAM15 (3 mg/kg ip) for 5 days before a 2 g/kg bolusglucose challenge. Control mice were injected daily with an equal volumeof carrier control (PEG400/5% DMSO, blue) n=4. (Right) After 8 days oftreatment mice were euthanized and tissues were weighed. Mice receivingBAM15 had less gonadal adipose tissue mass than vehicle controls. n=4.

FIG. 9. (also referred to as Example 2, FIG. 5) Flow chart of thescreens used to identify new mitochondrial uncouplers.

Example 3

FIGS. 10A.-10F. (also referred to as Example 3, FIG. 1) BAM15 is amitochondrial protonophore uncoupler with a broad effective range. (FIG.10A) Illustration of the proton motive force that generates ATP, and theuncoupling of nutrient oxidation from ATP production. (FIG. 10B) BAM15is a novel chemical uncoupler with no structural similarity to otherknown uncouplers, including FCCP. (FIG. 10C) Oxygen consumption rate(OCR) in L6 cells treated with BAM15 or FCCP at the indicatedconcentrations. (FIG. 10D) L6 cells were sequentially treated witholigomycin (Oligo, 1 μM), BAM15 or FCCP (Uncoupler, 1 μM), and antimycinA (10 μM) plus rotenone (1 μM) (A/R) as indicated by arrows. (FIG. 10E)TMRM-loaded L6 cells were treated with 10 μM BAM15 for 30 min prior toFACS analysis in the phycoerythrin (PE) channel. BAM15-treated cells areleft-shifted, indicating loss of mitochondrial membrane potential. (FIG.10F) Isolated mouse liver mitochondria respiring on pyruvate and malatein the presence of FCCP (5 μM) or BAM15 (5 μM) were treated sequentiallywith rotenone (4 μM), succinate (10 mM), antimycin A (4 μM), and theelectron donors TMPD (100 μM) and ascorbate (10 mM).*indicates p<0.05 bytwo-way ANOVA with Bonferroni's posttest. For (FIG. 10C) N=6-8 wells percondition from three separate experiments, (FIG. 10D) N=5 wells percondition over one experiment, (FIG. 10E) N=one representative fromthree separate experiments, (FIG. 10F) N=3 wells per condition from arepresentative of three separate experiments.

FIGS. 11A.-11H. (also referred to as Example 3, FIG. 2) BAM15 does notalter plasma membrane electrophysiology. (FIG. 11A) Representative wholecell voltage clamp recording from a L6 cell showing the holding current(at −70 mV) during 1 μM treatment of FCCP and BAM15. (FIG. 11B) Voltageclamp with 10 μM FCCP and BAM15. (FIG. 11C-11D) 10 currents wereelicited with a voltage ramp from −150 mV to +80 mV using 1 μMuncouplers in (FIG. 11C) and 10 μM uncouplers in (FIG. 11D). (FIG. 11E)Average data comparing the change in holding current caused by FCCP andBAM15 at 1 μM and 10 μM. (FIG. 11F) Average data comparing the change inconductance generated by either drug in the range of −130 mV to −60 mV.(FIG. 11G) Representative whole cell current clamp recording atconcentrations of 10 μM for FCCP and BAM15. (FIG. 11H) Average datacomparing the change in membrane potential by both drugs. For (FIG.11E), (FIG. 11F) and (FIG. 11H),* indicates p<0.05 by two-way ANOVA withBonferroni's posttest, n=7-9 cells per condition.

FIGS. 12A.-12C. (also referred to as Example 3, FIG. 3) BAM15 is lesscytotoxic than FCCP. (FIG. 12A) L6 and NMuLi cells were treated withincreasing concentrations of BAM15 or FCCP for 48 hrs and stained withcrystal violet. (FIG. 12B) L6 and NMuLi cells were treated with FCCP orBAM15 for 48 hrs and viewed with phase microscopy. (FIG. 12C) IC₅₀values of C2C12, NRVCs, L6, and NMuLi cells were calculated via MTTassay or crystal violet staining.

FIGS. 13A.-13C. (also referred to as Example 3, FIG. 4) BAM15 protectsagainst kidney ischemic-reperfusion injury. Male mice (8 wk old,C57BL/6) were treated with vehicle control (VC) or BAM15 at 1 or 5 mg/kgfor 1 hr prior to bilateral ischemia for 26 min followed by 48 hrs ofreperfusion. Sham-operated mice underwent a similar surgical procedure,but the renal pedicles were not clamped. (FIG. 13A) BAM15dose-dependently protected from elevated plasma creatinine levels at 24and 48 hrs following reperfusion. (FIG. 13B-13C) BAM15 pretreatmentdecreased acute proximal tubular necrosis as determined by histologicalanalysis of the kidney medulla 48 hrs following reperfusion. Arrowsindicate tubular necrosis.*indicates p<0.05 compared to vehicle controlby one-way ANOVA with Dunnett's posttest.

FIGS. 14A.-14C. (also referred to as Example 3, FIG. S1) Optimization ofcell density and FCCP positive control. L6 were seeded into a 96-wellBD-OBS microplate. Fluorescent BD-OBS signal intensity was recorded over50-60 min (1 read/min). (FIG. 14A) L6 were seeded at densities rangingfrom 0 to 1 million cells per well and immediately assayed forfluorescence increases with time. (FIG. 14B) L6 cells (5×10⁵ cells/wellin 100 μL) were treated with FCCP as a positive control to identify theoptimal concentration for screening. (FIG. 14C) Example of the hitresult for 5 μg/mL BAM15 using 5×10⁵ L6 cells/well and 10 μM FCCP as apositive control.

FIG. 15. (also referred to as Example 3, FIG. S2) The top 25 hits werescreened for intracellular ROS concentrations in L6 myoblasts. Cellswere incubated with CM-H₂DCFDA, a fluorescent indicator of ROS beforebeing treated with 5 μg/mL of the library compound used for screening.ROS is expressed in terms of percentage fluorescence of the vehiclecontrol (DMSO, white). Compounds that increased ROS over background wereeliminated (red dashed line). N=3.

FIGS. 16A.-16D. (also referred to as Example 3, FIG. S3) BAM15 has abroad effective range in multiple cell types. Oxygen consumption ratewas measured in L6 myoblasts (FIG. 16A), neonatal rat ventricularcardiomyocytes (FIG. 16B), C2C12 myoblasts (FIG. 16C), and NMuLihepatocytes (FIG. 16D) treated with a dose response of BAM15 or FCCP atthe concentrations indicated. The graphs on the right are magnified fromthe 0-1 μM doses outlined in the dashed box of the graph to theleft.*indicates p<0.05 by two-way ANOVA with Bonferroni's posttest.N=6-8 wells per condition from three separate experiments.

FIGS. 17A.-17D. (also referred to as Example 3, FIG. S4) BAM15 increasesextracellular acidification. Extracellular acidification rates weremeasured in L6 myoblasts (FIG. 17A), neonatal rat ventricularcardiomyocytes (FIG. 17B), C2C12 myoblasts (FIG. 17C), and NMuLihepatocytes (FIG. 17D) treated with a dose response of BAM15 versus FCCPas indicated. Error bars indicate SEM.*indicates p<0.05 by two-way ANOVAwith Bonferroni's posttest. N=6-8 wells per condition from threeseparate experiments.

FIGS. 18A.-18B. (also referred to as Example 3, FIG. S5) BAM15 increasesoxygen consumption in the presence of the ATP synthase inhibitoroligomycin. Oxygen consumption rate (OCR) was measured in L6 and NMuLicells following sequential treatment of 1 μM oligomycin, FCCP or BAM15(Uncoupler) at 2 μM (FIG. 18A) or 10 μM (FIG. 18B), and antimycin A (10μM) with rotenone (1 μM) (A/R).*indicates p<0.05 by two-way ANOVA withBonferroni' s posttest. N=3 wells/group. Error bars indicate SEM.

FIGS. 19A.-19D. (also referred to as Example 3, FIG. S6) Kidney monocytecontent including FIG. 19A) leukocytes and FIG. 19B) macrophages FIG.19C) neutrophils FIG. 19D) dendritic cells.*indicates p<0.05 by one wayANOVA with Dunnett' s posttest. N=3-6.

Example 4

FIG. 20. (also referred to as Example 4, FIG. 1) Pharmacokineticanalysis of an oral 5 mg/kg dose of BAM15 in mice.

DETAILED DESCRIPTION

Abbreviations and Acronyms

7-AAD-7-Aminoactinomycin D

AMPK-AMP-activated protein kinase

AntA-antimycin A

Ar-aryl

BAM15-also known as ST056388,(2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine,andN5,N6-bis(2-fluorophenyl)[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine

DNP—2,4-dinitrophenol

ECAR-extracellular acidification rate

ETC-electron transport chain

FCCP-carbonyl cyanide p-trifluoromethoxyphenylhydrazone

GTT-glucose tolerance test

HBA-hydrogen bond acceptor

HBD-hydrogen bond donor

HFD-high fat diet

HPβCD-hydroxypropyl β-cyclodextrin

OCR-oxygen consumption rate

PK-pharmacokinetic

PM-plasma membrane

ROS-reactive oxygen species

SAR-structure activity relationship

SILAC-stable isotope labeling of amino acids in cell culture

T2D-type 2 diabetes (also referred to as type II diabetes)

TCA-tricarboxylic acid cycle

TMPD-N,N,N′,N′-Tetramethyl-p-phenylenediamine

UCP-uncoupling protein

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below. Unlessdefined otherwise, all technical and scientific terms used herein havethe commonly understood by one of ordinary skill in the art to which theinvention pertains. Although any methods and materials similar orequivalent to those described herein may be useful in the practice ortesting of the present invention, preferred methods and materials aredescribed below. Specific terminology of particular importance to thedescription of the present invention is defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. For example, in oneaspect, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20%.

The terms “additional therapeutically active compound” or “additionaltherapeutic agent”, as used in the context of the present invention,refers to the use or administration of a compound for an additionaltherapeutic use for a particular injury, disease, or disorder beingtreated. Such a compound, for example, could include one being used totreat an unrelated disease or disorder, or a disease or disorder whichmay not be responsive to the primary treatment for the injury, diseaseor disorder being treated.

As used herein, the terms “administration of” and or “administering” acompound should be understood to mean providing a compound of theinvention or a prodrug of a compound of the invention to a subject inneed of treatment.

As used herein, an “agonist” is a composition of matter which, whenadministered to a mammal such as a human, enhances or extends abiological activity attributable to the level or presence of a targetcompound or molecule of interest in the subject.

As used herein, “alleviating a disease or disorder symptom,” meansreducing the severity of the symptom or the frequency with which such asymptom is experienced by a subject, or both.

As used herein, an “analog”, or “analogue” of a chemical compound is acompound that, by way of example, resembles another in structure but isnot necessarily an isomer (e.g., 5-fluorouracil is an analog ofthymine).

An “antagonist” is a composition of matter which when administered to amammal such as a human, inhibits a biological activity attributable tothe level or presence of a compound or molecule of interest in thesubject.

The term “antimicrobial agents” as used herein refers to anynaturally-occurring, synthetic, or semi-synthetic compound orcomposition or mixture thereof, which is safe for human or animal use aspracticed in the methods of this invention, and is effective in killingor substantially inhibiting the growth of microbes. “Antimicrobial” asused herein, includes antibacterial, antifungal, and antiviral agents.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above. When referring to acompound of the invention, and unless otherwise specified, the term“compound” is intended to encompass not only the specified molecularentity but also its pharmaceutically acceptable, pharmacologicallyactive analogs, including, but not limited to, salts, polymorphs,esters, amides, prodrugs, adducts, conjugates, active metabolites, andthe like, where such modifications to the molecular entity areappropriate.

The term “delivery vehicle” refers to any kind of device or materialwhich can be used to deliver compounds in vivo or can be added to acomposition comprising compounds administered to a plant or animal. Thisincludes, but is not limited to, implantable devices, aggregates ofcells, matrix materials, gels, etc.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

As used herein, an “effective amount” or “therapeutically effectiveamount” means an amount sufficient to produce a selected effect, such asalleviating symptoms of a disease or disorder. In the context ofadministering compounds in the form of a combination, such as multiplecompounds, the amount of each compound, when administered in combinationwith another compound(s), may be different from when that compound isadministered alone. Thus, an effective amount of a combination ofcompounds refers collectively to the combination as a whole, althoughthe actual amounts of each compound may vary. The term “more effective”means that the selected effect is alleviated to a greater extent by onetreatment relative to the second treatment to which it is beingcompared.

As used in the specification and the appended claims, the terms “forexample,” “for instance,” “such as,” “including” and the like are meantto introduce examples that further clarify more general subject matter.Unless otherwise specified, these examples are provided only as an aidfor understanding the invention, and are not meant to be limiting in anyfashion.

The terms “formula” and “structure” are used interchangeably herein.

As used herein, a “functional” molecule is a molecule in a form in whichit exhibits a property or activity by which it is characterized. Afunctional enzyme, for example, is one that exhibits the characteristiccatalytic activity by which the enzyme is characterized.

As used herein, “homology” is used synonymously with “identity.”

The term “inhibit,” as used herein, refers to the ability of a compoundof the invention to reduce or impede a described function, such ashaving inhibitory sodium channel activity. Preferably, inhibition is byat least 10%, more preferably by at least 25%, even more preferably byat least 50%, and most preferably, the function is inhibited by at least75%. The terms “inhibit”, “reduce”, and “block” are used interchangeablyherein.

As used herein “injecting or applying” includes administration of acompound of the invention by any number of routes and means including,but not limited to, topical, oral, buccal, intravenous, intramuscular,intra arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the identified compound invention or be shipped togetherwith a container which contains the identified compound. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the instructional material and the compound beused cooperatively by the recipient.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc. and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present. Such assays are coupled with processes or systems tostore and process information and to help quantify levels, signals, etc.and to digitize the information for use in comparing levels.

The term, “mitochondrial uncoupling”, also referred to as “uncoupling”,refers to the process whereby protons enter the mitochondrial matrix viaa pathway independent of ATP synthase and thereby uncouple nutrientoxidation from ATP production. This process can be pharmacologicallyinduced by small molecule mitochondrial protonophores, which directlyshuttle protons across the mitochondrial inner membrane into the matrix.The primary pathway for energy production in aerobic cells involves theoxidation of nutrients (including fats, carbohydrates, and amino acids)in mitochondria, which promotes the efflux of protons out of themitochondrial matrix. This process creates a pH and electrochemicalgradient across the mitochondrial inner membrane. Protons normallyre-enter the mitochondrial matrix via ATP synthase, which results in ATPproduction. Protons can also re-enter the mitochondrial matrix viapathways independent of ATP synthase, which ‘uncouples’ nutrientoxidation and proton efflux from ATP production.

The term “modulate”, as used herein, refers to changing the level of anactivity, function, or process. The term “modulate” encompasses bothinhibiting and stimulating an activity, function, or process.

The term “per application” as used herein refers to administration of acompositions, drug, or compound to a subject.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “preventive” or “prophylactic” treatment is a treatment administeredto a subject who does not exhibit signs, or exhibits only early signs,of a disease or disorder. A prophylactic or preventative treatment isadministered for the purpose of decreasing the risk of developingpathology associated with developing the disease or disorder.

A “prodrug” refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug, or may demonstrate increased palatability or beeasier to formulate.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

A “sample,” as used herein, refers preferably to a biological samplefrom a subject or assay materials, including, but not limited to, normaltissue samples, diseased tissue samples, biopsies, blood, saliva, feces,semen, tears, cell extracts, and urine. A sample can also be any othersource of material obtained from a subject which contains cells,tissues, or fluid of interest. A sample can also be obtained from cellor tissue culture.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered and used for comparing results when administeringa test compound, or it can be a standard parameter or function which ismeasured to obtain a control value when measuring an effect of an agentor compound on a parameter or function. Standard can also refer to an“internal standard”, such as an agent or compound which is added atknown amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured. Internal standards are often a purified marker of interestwhich has been labeled, such as with a radioactive isotope, allowing itto be distinguished from an endogenous marker.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

As used herein, a “subject in need thereof” is a patient, animal,mammal, or human, who will benefit from the method of this invention.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. A “prophylactic” treatment is a treatment administered to asubject who does not exhibit signs of a disease or exhibits only earlysigns of the disease for the purpose of decreasing the risk ofdeveloping pathology associated with the disease.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

As used herein, the term “treating” includes prophylaxis of the specificdisease, disorder, or condition, or alleviation of the symptomsassociated with a specific disease, disorder, or condition and/orpreventing or eliminating said symptoms.

As used herein, the term “wound” relates to a physical tear, break, orrupture to a tissue or cell layer. A wound may occur by any physicalinsult, including a surgical procedure or as a result of a disease,disorder condition.

Chemical Definitions

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “alkyl or C₁-C₁₀ alkyl,” as used herein, represents a branchedor linear alkyl group having from one to six carbon atoms. TypicallyC₁-C₁₀ alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl,hexyl, heptyl, octyl, and the like.

The term “alkenyl or C₂-C₁₀ alkenyl,” as used herein, represents anolefinically unsaturated branched or linear group having from 2 to 10carbon atoms and at least one double bond. Examples of such groupsinclude, but are not limited to, 1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl, and the like.

The term “alkynyl or C₂-C₁₀ alkynyl,” refers to an unsaturated branchedor linear group having from 2 to 10 carbon atoms and at least one triplebond. Examples of such groups include, but are not limited to,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C₈ cycloalkyl,” represents cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

As used herein the term “aryl” refers to a mono or bicyclic C₅-C₁₀carbocyclic ring system having one or two aromatic rings including, butnot limited to, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl,indenyl, and the like.

As used herein “optionally substituted aryl” includes aryl compoundshaving from zero to four substituents, and a substituted aryl includesaryl compounds having one to three substituents, wherein thesubstituents include groups such as, for example, alkyl, halo or aminosubstituents.

The term “arylalkyl” refers to any aryl group which is attached to theparent moiety via the alkyl group, e.g., aryl(C₁-C₈ alkyl). Thus, theterm (C₅-C₆ aryl)(C₅-C₈ alkyl) refers to a five or six membered aromaticring that is attached to the parent moiety via the C₅-C₈ alkyl group.

The term “heterocyclic group” refers to an optionally substituted mono-or bicyclic carbocyclic ring system containing from one to threeheteroatoms wherein the heteroatoms are selected from the groupconsisting of oxygen, sulfur, and nitrogen.

As used herein the term “heteroaryl” refers to an optionally substitutedmono- or bicyclic carbocyclic ring system having one or two aromaticrings containing from one to three heteroatoms and includes, but is notlimited to, furyl, thienyl, pyridyl and the like.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The compounds of the present invention can contain one or moreasymmetric centers in the molecule. In accordance with the presentinvention any structure that does not designate the stereochemistry isto be understood as embracing all the various optical isomers, as wellas racemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example, the following structure:

is understood to represent a mixture of the structures:

as well as

The terms 16:0, 18:0, 18:1, 20:4 or 22:6 hydrocarbon refers to abranched or straight alkyl or alkenyl group, wherein the first integerrepresents the total number of carbons in the group and the secondinteger represent the number of double bonds in the group.

Embodiments

Mitochondria regulate cellular metabolism and play an important role inthe pathogenesis of some of the most prevalent human diseases includingobesity, cancer, diabetes, neurodegeneration, and heart disease. Thecompounds of the invention, including BAM15, are useful for treating andpreventing these diseases and disorders and other described herein, aswell as others where a mitochondrial uncoupler is useful.

BAM15 is in the public domain. It is arbitrarily named BAM15 herein. ItsIUPAC name is5-N,6-N-bis(2-fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine,it is compound number ST056388 from Timtec. The library it came from isthe Timtec ApexScreen 5040.

Many anti-diabetes drugs such as insulin-sensitizers promote glucoseclearance from the blood by effectively ‘pushing’ glucose into nutrientoverloaded tissues; however, in contrast to this approach our strategyis aimed at reducing cellular nutrient stores so that tissues will‘pull’ glucose from the circulation. The present method is modeled afterexercise and calorie restriction interventions which also reducecellular nutrient stores to improve glycemia and insulin sensitivity.The proof of principle is validated in humans treated with themitochondrial uncoupler 2,4-dinitrophenol (DNP). DNP decreases adiposityand improves metabolism in humans; however, it also has a very narrowtherapeutic window and was removed from FDA approval in 1938. Otheranti-diabetes drugs including agonists of thyroid hormone and inhibitorsof 11-β hydroxysteroid dehydrogenase type 1 have off-target effects ofincreased energy expenditure that may mediate some of the protectiveeffects of these compounds. Nevertheless, there are no drugs have beenspecifically targeted for increased energy expenditure.

The compound BAM15 shows promising insulin sensitizing effects incultured cells and mice.

One of the earliest defects observed in Type 2 diabetes is reducedinsulin sensitivity, or insulin resistance, and so restoring thisprocess is a major aim of many therapeutic strategies. Thenon-pharmacological interventions of exercise and calorie restrictionare very effective reversers of T2D; however they require strict andvigorous adherence to protocol and receive poor patient compliance. Assuch, pharmacological intervention in diabetes is necessary. Most, ifnot all, current anti-diabetes drugs were identified based on theirblood glucose-lowering properties; however one problem with thisapproach is that it does not consider the consequences of glucoseoverload into peripheral tissues. In an effort to develop new approachesfor intervention in diabetes we have developed a novel drug screen thatis modeled upon the mechanisms of action of diet and exercise; includingcellular nutrient consumption, amplified antioxidant defense, andinsulin sensitivity.

Useful compounds of the invention include, but are not limited to,BAM15, BAM8, BAM9, FCCP, and 2,4-dinitrophenol, as well as biologicallyactive analogs and derivatives thereof.

In one embodiment, a compound of the invention has the general formula:

as well as active analogs and derivatives thereof.

In one aspect, R₁-R₁₀ are all independently optional, and when presentare each independently selected from the group consisting of halogen(halo). In one aspect, the halogen is F, Cl, Br, or I. In one aspect itis F. In one aspect, each of R₁-R₁₀ is independently selected from thegroup consisting of: H, halogen, hydroxy, acyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol,sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acidsidechain, and amino acid (each group can be optionally substituted), ora pharmaceutically acceptable salt or prodrug thereof. In one aspect,the halogen is F, Cl, Br, or I. In one aspect it is F.

In one embodiment, a compound of the invention has the general formulaII:

as well as active analogs and derivatives thereof. In one aspect, R₁-R₂are independently optional, and when present are each independentlyselected from the group consisting of halogen (halo). In one aspect,each is independently selected from the group consisting of: H, halogen,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, and amino acid (eachgroup can be optionally substituted), or a pharmaceutically acceptablesalt or prodrug thereof. In one aspect, the halogen is F, Cl, Br, or I.In one aspect it is F.

One of ordinary skill in the art will appreciate that not allconfigurations need to be effective or as effective as other compoundsof the genus.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase) and how to determine compound activity using thestandard tests described herein, or using other similar tests which arewell known in the art.

In one embodiment, the present invention provides compositions andmethods for preventing or treating a disease, disorder, or condition,comprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, optionallyat least one additional therapeutic agent, and an effective amount of atleast one compound having a structure of Formula I or Formula II. In oneaspect, the disease, disorder or condition is selected from the groupconsisting of ischemia reperfusion injury, hyperinsulinemia,hyperlipidemia, glycemia, glucose tolerance, insulin sensitivity,adiposity, insulin resistance, obesity, diabetes, cancer,neurodegeneration, heart disease, heart failure, Parkinson's disease,aging, and disorders standing to benefit from increased energyexpenditure. In one aspect, the compound is compound is a mitochondrialuncoupler. In one aspect, the diabetes is type II diabetes. In oneaspect, the ischemia reperfusion injury is kidney ischemia reperfusioninjury or cardiac ischemia reperfusion injury. In one aspect, the methodreduces reperfusion-induced mitochondrial oxidative stress andmitochondrial fragmentation.

Compounds of the invention can be administered to a subject at varioustimes, dosages, and more than once, depending on, for example, the age,sex, health, and weight of the subject, as well as on the particulardisease, disorder, or condition to be treated or prevented. In oneaspect, a compound is administered at a dosage ranging from about 0.1mg/kg to about 50 mg/kg body weight. In another aspect, the compound isadministered at a dosage ranging from about 0.5 mg/kg to about 25 mg/kgbody weight. In yet another aspect, the compound is administered at adosage ranging from about 1.0 mg/kg to about 5.0 mg/kg body weight. Inone aspect, about 3.0 mg/kg is administered. In another aspect, about5.0 mg/kg is administered. In another aspect, the compound isadministered as a unit dose ranging from about 10 mg to about 500mg/unit dose. In one aspect, the compound is administered more thanonce. In one aspect, the compound is a mitochondrial protonophoreuncoupler lacking protonophore activity at the plasma membrane.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently basic compound such asan amine with a suitable acid affording a physiologically acceptableanion. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts of organic (e.g.,carboxylic) acids can also be made.

Processes for preparing compounds of a generic formula of the invention,such as formulas I or II, or for preparing intermediates useful forpreparing compounds of formula I or other formulas of the invention areprovided as further embodiments of the invention or are known in theart. Intermediates useful for preparing compounds of formula I or otherformulas are also provided as further embodiments of the invention.

Useful compounds of the invention include, but are not limited to:

BAM15 is further described at the national library of medicine websitein the “pubchem” section, where it is referred to as compound ID 565708.Properties of BAM15 include: Molecular Weight: 340.287006 [g/mol] andMolecular Formula: C₁₆H₁₀F₂N₆O. Its chemical names are(2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amineandN5,N6-bis(2-fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine(its IUPAC name).

Other useful compounds for aspects of the invention include FCCP and2,4-dinitrophenol, having the following structures:

In one embodiment, one or more compounds of the invention areadministered to a subject in need thereof, including, but not limitedto, a Type II diabetic. In one aspect, a pharmaceutical compositioncomprising an effective amount of at least one compound of the inventionis administered to the subject.

In one aspect, administration of a compound of the invention improvesglucose tolerance. In one aspect, BAM15 improves glucose tolerance. Inone aspect, administration of a compound of the invention providesprotection from high fat diet-induced glucose intolerance. In oneaspect, a compound of the invention is an agonist of energy expenditureand increases oxygen consumption without ROS production and activatesAMPK without depletion of ATP.

In one aspect, a compound of the invention increases cellular oxygenconsumption.

In one embodiment, a compound of the invention reverses insulinresistance. In one aspect, the compound reverses or treatshyperinsulinemia. In one aspect, the compound reverses or treatshyperlipidemia. In one aspect, a compound of the invention improvesglucose tolerance. In one aspect, a compound of the invention improvesglucose tolerance in a subject on a high fat diet. In one embodiment, acompound of the invention is useful for increasing cellular oxygenconsumption. In one embodiment, a compound of the invention is useful asan anti-diabetic.

In one aspect, administration of a compound of the invention to asubject in need thereof will cause improvements in blood lipid profiles,glucose tolerance, leanness, and insulin sensitivity. In one aspect,improvements in blood lipid profiles, glucose tolerance, leanness, andinsulin sensitivity occur without hypophagia. In one aspect,improvements in blood lipid profiles, glucose tolerance, leanness, andinsulin sensitivity occur without hyperinsulinemia. In one aspect,improvements in blood lipid profiles, glucose tolerance, leanness, andinsulin sensitivity occur without hypophagia or hyperinsulinemia. Theinvention therefore encompasses the use of BAM15 and other compounds ofthe invention having similar activity for use in treating and preventingobesity.

A compound of the invention, such as BAM15, has certain properties thatcan be tested for and identified in other compounds using the methods ofthe invention. For example, a compound of the invention has themeasurable properties required of a mitochondrial protonophore uncouplerwhen subjected to a series of biochemical assays such as the abilityto: 1) stimulate OCR when ATP synthase is inhibited; 2) depolarize themitochondrial inner membrane; 3) stimulate respiration in isolatedmitochondria; and 4) increase OCR without donating electrons to theelectron transport chain.

In one aspect, a compound of the invention comprises a molecular weightbetween 205-370, HBA<5, HBD<3, 1-3 rings, and a calculated Log S of>10-3.

The present invention further provides compositions and methods foridentifying compounds comprising the activity described herein. Thenovel screening assay is modeled upon the mechanisms of action of dietand exercise, including cellular nutrient composition, amplifiedantioxidant defense, and insulin sensitivity. In one aspect, the assayis exemplified by FIG. 1.

The present invention further encompasses compounds identified by themethods of the invention.

As described herein, the compositions of the present invention comprise,as an active agent, compounds having the structure of any of theformulas disclosed herein in a pharmaceutically acceptable form. Ifdesired, the compositions may further comprise one or more additionalactive agents. Where it is appropriate, any of the active agents may beadministered in the form of the compound per se, and/or in the form of asalt, polymorph, ester, amide, prodrug, derivative, or the like,provided the salt, polymorph, ester, amide, prodrug or derivative issuitable pharmacologically. Where it is appropriate, salts, esters,amides, prodrugs and other derivatives of the active agents may beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by J. March,Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992). For any active agents that mayexist in enantiomeric forms, the active agent may be incorporated intothe present compositions either as the racemate or in enantiomericallyenriched form.

The values provided herein for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents. Thedisclosed compounds include compounds of the specific Formulas recitedherein having any combination of the exemplary values, preferred values,and more preferred values described herein.

In one embodiment, at least one of the compounds being administered isadministered at least once a day. In one aspect, a compound isadministered more than once. In one aspect, it is administered at leasttwice a day. In another embodiment, it is administered at least once aweek. In yet another embodiment, it is administered at least once amonth.

The invention further provides pharmaceutical compositions comprisingcompounds of the invention. The pharmaceutical composition may compriseone or more compounds of the invention, and biologically active analogs,homologs, derivatives, modifications, and pharmaceutically acceptablesalts thereof, and a pharmaceutically acceptable carrier. In oneembodiment, the compounds are administered as a pharmaceuticalcomposition.

The route of administration can vary depending on the type of compoundbeing administered. In one aspect, the compounds are administered viaroutes such as oral, topical, rectal, intramuscular, intramucosal,intranasal, inhalation, ophthalmic, and intravenous.

The present invention further provides for administration of a compoundof the invention as a controlled-release formulation.

In one embodiment, the present invention provides administering at leastthree compounds, wherein at least three of the compounds are topiramate,ondansetron, and naltrexone.

In one embodiment, the present invention provides compositions andmethods for treating alcohol-related diseases and disorders usingpharmaceutical compositions comprising effective amounts of topiramate,ondansetron, and naltrexone.

The dosage of the active compound(s) being administered will depend onthe condition being treated, the particular compound, and other clinicalfactors such as age, sex, weight, and health of the subject beingtreated, the route of administration of the compound(s), and the type ofcomposition being administered (tablet, gel cap, capsule, solution,suspension, inhaler, aerosol, elixir, lozenge, injection, patch,ointment, cream, etc.). It is to be understood that the presentinvention has application for both human and veterinary use.

Processes for preparing compounds of any of the formulas of theinvention or for preparing intermediates useful for preparing compoundsof any of the formulas of the invention are provided as furtherembodiments of the invention. Intermediates useful for preparingcompounds of formula I are also provided as further embodiments of theinvention.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently basic compound such asan amine with a suitable acid affording a physiologically acceptableanion. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts of carboxylic acids canalso be made.

Processes for preparing compounds of any of the formulas of theinvention are provided as further embodiments of the invention and areillustrated by the following procedures in which the meanings of thegeneric radicals are as given above unless otherwise qualified.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,-ketoglutarate, and-glycerophosphate. Suitable inorganic salts may alsobe formed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of any of the formulas of the invention can be formulatedas pharmaceutical compositions and administered to a mammalian host,such as a human patient in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

It will be appreciated that compounds of the invention can beadministered using various kinds of delivery systems and media.Furthermore, compounds of the invention can be administered incombination with other therapeutic agents and compounds and can be usedwith other kinds of treatments.

In one embodiment, the present compounds may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, theactive compound may be combined with one or more excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, and the like. Such compositionsand preparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate, and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze-drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I or formula II to the skin are knownto the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157)and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the formulas of the invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of formula I in a liquidcomposition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

For example, in one embodiment relating to oral administration tohumans, a dosage of between approximately 0.1 and 300 mg/kg/day, orbetween approximately 0.5 and 50 mg/kg/day, or between approximately 1and 10 mg/kg/day, is generally sufficient, but will vary depending onsuch things as the disorder being treated, the length of treatment, theage, sex, weight, and/or health of the subject, etc. In one aspect, aunit dose is used. In one aspect, the unit dose is supplied in asyringe. The combinations of drugs can be administered in formulationsthat contain all drugs being used, or the drugs can be administeredseparately. In some cases, it is anticipated that multiple doses/timesof administration will be required or useful. Additionally, for sometreatment regimens, at least two compounds will be used. In one aspect,at least three compounds will be administered. The present inventionfurther provides for varying the length of time of treatment.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day. The compound isconveniently administered in unit dosage form; for example, containing 5to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mgof active ingredient per unit dosage form.

Ideally, when the active ingredient needs to enter circulation and bedelivered via blood, the active ingredient, in one embodiment, should beadministered to achieve peak plasma concentrations of the activecompound of from about 0.5 to about 75 μM preferably, about 1 to 50 μMmost preferably, about 2 to about 30 μM. This may be achieved, forexample, by the intravenous injection of a 0.05 to 5% solution of theactive ingredient, optionally in saline, or orally administered as abolus containing about 1-100 mg of the active ingredient. Desirableblood levels may be maintained by continuous infusion to provide about0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

In another embodiment, a formulation of the invention can be impregnatedinto a dressing material (or otherwise contained or encompassed by thedressing material). The dressing material is a pharmaceuticallyacceptable fabric. It can be, for example, gauze or any other type ofmedical fabric or material that can be used to cover a wound and/or tokeep a therapeutic agent or composition in contact with a patient.

The composition of the invention can further comprise additionaltherapeutic additives, alone or in combination (e.g., 2, 3, or 4additional additives). Examples of additional additives include but arenot limited to: (a) antimicrobials, (b) steroids (e.g., hydrocortisone,triamcinolone); (c) pain medications (e.g., aspirin, an NSAID, and alocal anesthetic); (d) anti-inflammatory agents; and (e) combinationsthereof.

Non-synthetic matrix proteins like collagen, glycosaminoglycans, andhyaluronic acid, which are enzymatically digested in the body, areuseful for delivery (see U.S. Pat. Nos. 4,394,320; 4,472,840; 5,366,509;5,606,019; 5,645,591; and 5,683,459) and are suitable for use with thepresent invention. Other implantable media and devices can be used fordelivery of the compounds of the invention in vivo. These include, butare not limited to, sponges, such as those from Integra, fibrin gels,scaffolds formed from sintered microspheres of polylactic acid glycolicacid copolymers (PLAGA), and nanofibers formed from native collagen, aswell as other proteins. The compounds of the present invention can befurther combined with growth factors, nutrient factors, pharmaceuticals,calcium-containing compounds, anti-inflammatory agents, antimicrobialagents, or any other substance capable of expediting or facilitatingbone or tissue growth, stability, and remodeling.

The compositions of the present invention can also be combined withinorganic fillers or particles. For example for use in implantablegrafts the inorganic fillers or particles can be selected fromhydroxyapatite, tri-calcium phosphate, ceramic glass, amorphous calciumphosphate, porous ceramic particles or powders, mesh titanium ortitanium alloy, or particulate titanium or titanium alloy.

Examples of other antimicrobial agents that can be used in the presentinvention include, but are not limited to, isoniazid, ethambutol,pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones,ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin,dapsone, tetracycline, erythromycin, cikprofloxacin, doxycycline,ampicillin, amphotericine B, ketoconazole, fluconazole, pyrimethamine,sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone,paromomycin, diclarazaril, acyclovir, trifluorouridine, foscarnet,penicillin, gentamicin, ganciclovir, iatroconazole, miconazole,Zn-pyrithione, and silver salts, such as chloride, bromide, iodide, andperiodate.

In one embodiment, the compounds of the invention can first beencapsulated into microcapsules, microspheres, microparticles,microfibers, reinforcing fibers and the like to facilitate mixing andachieving controlled, extended, delayed and/or sustained release andcombined other agents or drugs. Encapsulating the biologically activeagent can also protect the agent against degradation during formation ofthe composite of the invention.

In another embodiment of the invention, the compound is controllablyreleased into a subject when the composition of the invention isimplanted into a subject, due to bioresorption relying on the time scaleresulting from cellular remodeling. In one aspect, the composition maybe used to replace an area of discontinuity in the tissue. The area ofdiscontinuity can be the result of trauma, a disease, disorder, orcondition, surgery, injury, etc.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor its designated use. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thecomposition or be shipped together with a container which contains thecomposition. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the composition be used cooperatively by the recipient.

The method of the invention includes a kit comprising a compoundidentified in the invention and an instructional material whichdescribes administering the compound or a composition comprising thecompound to a cell or a subject to any target of interest, such as asurface. This should be construed to include other embodiments of kitsthat are known to those skilled in the art, such as a kit comprising a(preferably sterile) solvent suitable for dissolving or suspending thecomposition of the invention prior to administering the compound to acell or a subject. Preferably the subject is a human.

In accordance with the present invention, as described above or asdiscussed in the Examples below, there can be employed conventionalchemical, cellular, histochemical, biochemical, molecular biology,microbiology, and in vivo techniques which are known to those of skillin the art. Such techniques are explained fully in the literature.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention.

The invention is now described with reference to the following Examplesand Embodiments. Without further description, it is believed that one ofordinary skill in the art can, using the preceding description and thefollowing illustrative examples, make and utilize the present inventionand practice the claimed methods. The following working examplestherefore, are provided for the purpose of illustration only andspecifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure. Therefore, the examples should be construedto encompass any and all variations which become evident as a result ofthe teaching provided herein.

EXAMPLES

General Methods

Materials

The ApexScreen 5040 library was purchased from TimTec (Newark, D E) witheach chemical compound supplied at a concentration of 1 μg/μL indimethyl sulfoxide (DMSO). 96-well Becton Dickinson (BD) OxygenBiosensor (OBS) microplates were obtained from BD Biosciences (Bedford,Mass.). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) andCM-H₂DCFDA were purchased from Sigma-Aldrich and Molecular Probes,Invitrogen (Carlsbad, Calif.), respectively.

Oxygen Consumption Assay

L6 myoblasts were grown to confluence, washed with PBS, trypsinized, andthen seeded into a 96-well BD-OBS microplate at a density of 500,000cells/well in 100 μL of L6 growth medium. Cells were incubated with 0.5%(v/v) library compound or vehicle control (DMSO) and fluorescenceintensity recorded over 45-90 min (1 read/min) at 37° C. by a SpectraMaxM5 dual-monochromator microplate reader (Molecular Devices, CA) using abottom-read configuration and with the excitation and emission filtersset at 485 nm and 630 nm, respectively. The mitochondrial uncouplingagent FCCP was used as positive control for uncoupling. Fluorescencedata were recorded on SoftMax Pro (version 4.8) software and exported toMicrosoft Excel for further analysis. Compounds which increased oxygenconsumption by >20% relative to control cells were selected forsecondary screening.

ROS Production Assay

L6 myoblasts were seeded into black-walled clear-bottom 96-wellmicroplates in L6 growth media and grown to confluence. Cells were thenwashed twice with PBS and co-incubated with 7.5 μM CM-H₂DCFDA and 0.5ng/μL of each hit compound or vehicle control (DMSO) in KRP buffer (136mM NaCl, 4.7 mM KCl, 10 mM NaPO₄, 0.9 mM MgSO₄, 0.9 mM CaCl₂, pH 7.4)supplemented with 25 mM D-glucose at 37° C. in 5% CO₂/95% air for 1 hr.100 nM H₂O₂ was used as a positive control for ROS production. Followingincubation, cells were washed three times with PBS to remove excessprobe. Cells were then covered with 100 μL/well PBS and fluorescenceintensity measured by a Tecan Infinite® M200 microplate reader (TecanGroup Ltd., Switzerland) using a top-read configuration and with theexcitation and emission filters set at 495±9 nm and 530±20 nm,respectively. Fluorescence data were recorded on Magellan (version 6.4)software and exported to Microsoft Excel for subsequent analysis. Havingsubtracted the background fluorescence (that emitted from a well whichdid not receive the CM-H₂DCFDA probe) from each well, ROS production wasexpressed in terms of percentage fluorescence of the vehicle control foreach condition. Compounds which increased ROS levels by greater than 20%were eliminated.

Measurements of Oxygen Consumption and Extracellular Acidification inWhole Cells

Oxygen consumption rate (OCR) and extracellular acidification rate(ECAR) were measured using a Seahorse XF-24 Flux Analyzer (SeahorseBiosciences, North Billerica, Mass.). NMuLi, C2C12, and L6 cells wereseeded in a Seahorse 24-well tissue culture plate at a density of3.5×10⁴ cells/well, and isolated cardiomyocytes at a density of 4×10⁴cells/well. The cells were then allowed to adhere overnight. Prior tothe assay, the media was changed to unbuffered DMEM containing pyruvateand glutamine (Gibco #12800-017, pH=7.4 at 37° C.) and the cells wereequilibrated for 30 mins at 37° C. without CO₂. Compounds were injectedduring the assay and OCR and ECAR were measured using 2 min measurementperiods. 2-3 wells were used per condition and averaged over threeplates (n=6-9). Statistical significance was determined by two-way ANOVAwith Bonferroni's posttest.

Mitochondria Isolation

Mitochondria were isolated from the livers of male C57BL/6 mice. Micewere sacrificed via cervical dislocation. Livers were removed, mincedwith scissors, and immediately placed in 1 mL ice-cold isolation medium(250 mM sucrose, 10 mM Tris-HCl, 1 mM EGTA, 1% fatty acid free BSA, pH7.4). The tissue was homogenized using four strokes of an automatedPotter-Elvehjem tissue homogenizer. After adding 4 mL of isolationmedium the homogenate was centrifuged at 800×g for 10 min at 4° C. Thesupernatant was then divided into four 2 mL Eppendorf tubes andcentrifuged at 12,000×g for 10 min at 4° C. The supernatant was removedand any white debris was aspirated from the brown mitochondria pellet.The pellets were then combined in 1 mL isolation medium and centrifugedat 10,000×g for 10 min. The supernatant and any white debris wereremoved and the mitochondria were resuspended in 1 mL mitochondrialassay solution (MAS, 70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mMMgCl₂, 2 mM HEPES, 1 mM EGTA, 0.2% fatty acid free BSA, pH 7.2).

Electron Flow Assay

Electron flow assays were performed using the methods described inRogers et al. (19). Briefly, 5 μg of mitochondrial protein in MAS wasloaded into a Seahorse 24-well tissue culture plate and centrifuged at2000×g for 15 min at 4° C. Prior to the assay, mitochondria wereincubated at 37° C. for 10 mins in MAS containing 10 mM pyruvate, 2 mMmalate, and 5 μM BAM15 or FCCP. Rotenone (2 μM), Succinate (10 mM),Antimycin A (4 μM), and N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD,100 μM) plus Ascorbate (10 mM) were added sequentially over time. N=3wells/plate of a representative of 3 plates.

Mitochondrial Titration Assays

Mouse liver mitochondria were isolated and respiration was measuredaccording to Rogers et al (19). Oxygen consumption was measured using aSeahorse XF96 Flux Analyzer on mitochondria respiring on pyruvate (10mM) and malate (2 mM) or Succinate (10 mM) and Rotenone (2 μM).

Mitochondrial Membrane Potential

L6 cells were incubated with the fluorescent indicator of mitochondrialmembrane potential TMRM (250 nM) or DMSO (1%) control for 30 mins. Thecells were then centrifuged for 10 min at 700× g and resuspended inculture media (alpha-MEM supplemented with 10% fetal calf serum) at aconcentration of 1×10⁵ cells/mL and treated with 10 μM BAM15/FCCP orDMSO (0.1%) for 30 min prior to flow cytometric analysis. n=3.

Plasma Membrane Electrophysiology

In preparation for recording, L6 cells were plated ontopoly-L-lysine-coated glass coverslips and returned to the incubator toadhere for at least 1 hr prior to use. Cells were used within 1 day ofplating. Whole cell recordings were performed at room temperature with3-5 MΩ Sylgard-coated borosilicate glass patch pipettes and an Axopatch200B amplifier (Molecular Devices). The internal solution contained 120mM KCH₃SO₃, 4 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 10 mM HEPES, 10 mMEGTA, 3 mM ATP-Mg and 0.3 mM GTP-Tris (pH 7.2). The bath solution wascomposed of 140 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPESand 10 mM glucose (pH 7.3) and was flowed over the cells atapproximately 2 ml/min. For voltage clamp experiments, cells were heldat-70 mV and a 750 msec ramp from −150 mV to +80 mV was applied at 10sec intervals using pCLAMP software and a Digidata 1322A digitizer(Molecular Devices). Conductance measurements were taken between −130 mVand −60 mV. For current clamp experiments, cells were recorded either atthe resting membrane potential or with current injection to reach apotential of approximately −70 mV.

Cytotoxicity

Cells were seeded into 96 well plates at a density of 5,000 cells/wellfor NMuLi, L6 and C2C12 cells and 10,000 cells/well for primary rat leftventricular cardiomyocytes. Cells were incubated overnight at 37° C.prior to drug treatment. Drugs were diluted in cell culture medium (10%fetal calf serum in Dulbecco's Modified Eagle Medium) (Gibco LifeTechnologies, Grand Island, N.Y., USA) and added to each well at theindicated concentrations. Cell viability was measured 48 h later using3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution(MTT) (Amresco, Solon, Ohio, USA) or crystal violet staining (0.5% w/vin 50% methanol). Absorbance was measured using a SpectraMax M5 platereader (Molecular Devices, Sunnyvale, Calif., USA). Cell viability ofdrug-treated cells is displayed as a percentage of control cells i.e.cells with equivalent concentrations of the vehicle, dimethylsulfoxide(DMSO). The final concentration of DMSO exposed to the cells was no morethan 0.1% (v/v) for the duration of the experiment.

Renal Ischemic Reperfusion Injury

All animals were handled and procedures were performed in adherence tothe National Institutes of Health Guide for the Care and Use ofLaboratory Animals, and all protocols were approved by the University ofVirginia Institutional Animal Care and Use Committee. Male mice (8 wkold, C57BL/6, from the National Cancer Institute, Frederick, Md.) wereanesthetized with a mixture (i.p.) of ketamine (120 mg/kg), xylazine (12mg/kg), and atropine (0.324 mg/kg) and were subjected to bilateralischemic reperfusion injury (26 min ischemia, then 24 h or 48 hreperfusion) as previously described (24). During the surgery, mousecore temperature was maintained at 34-36° C. with a heating pad; duringthe recovery and reperfusion period, mice were housed in a warmingincubator with ambient temperature at 30-32° C. Control, sham-operatedmice underwent a similar procedure, but the renal pedicles were notclamped. Mice were i.p. injected with BAM15 at 1 or 5 mg/kg, 1 h beforekidney IR. Vehicle mice were also injected with the same solution BAM15was prepared with (3% DMSO in 50% PEG400).

Assessment of Kidney Function and Histology

Plasma creatinine, as a measure of kidney function, was determined usinga colorimetric assay according to the manufacturer's protocol(Sigma-Aldrich). For histology, kidneys were fixed overnight in 0.2%sodium periodate/1.4% DL-lysine/4% paraformaldehyde in 0.1 M phosphatebuffer (pH 7.4) and embedded in paraffin. Kidneys were prepared for H&Estaining as previously described (3) and viewed by light microscopy(Zeiss Axioskop). Photographs were taken and brightness/contrastadjustment was made with a SPOT RT camera (software version 3.3;Diagnostic Instruments). Acute tubular necrosis was assessed aspreviously described (25). Stained kidney sections were scored in ablinded manner. The score was based on the percentage of outer medullatubules with pink casts on the inside, which is a marker of tubularnecrosis. The scoring system was as follows: 1 (<10%), 2 (10 to 25%), 3(25 to 75%), and 4 (>75%).

Kidney FACS Analysis

Flow cytometry was used to analyze kidney leukocyte content. In brief,kidneys were extracted, minced, digested, and passed through a filterand a cotton column as described (26). After blocking non-specific Fcbinding with anti-mouse CD16/32 (2.4G2), fresh kidney suspensions wereincubated with fluorophore-tagged anti-mouse CD45 (30-F11) to determinetotal leukocyte cell numbers. CD45-labeled samples were further used forlabeling with different combinations of anti-mouse F4/80 (BM8), GR-1(Ly6G), CD11b, CD11c, IA (MHCII). 7-Aminoactinomycin D (7-AAD; BDBiosciences) was added 15 min before analyzing the sample to separatelive from dead cells. Flow cytometry data acquisition was performed on aFACSCalibur (Becton Dickinson). Data were analyzed by FlowJo software9.0 (Tree Star).

Example 1

We recently screened a diversity chemical library in rat L6 skeletalmuscle cells for energy expenditure agonists with antioxidant propertiesthat improved insulin sensitivity. More than 5,000 molecules were passedthrough the first screen for energy expenditure agonists. This produced25 positive hits, a hit rate of ˜0.5%. Our secondary screen for ROSproduction ruled out false positives that increased oxygen consumptionvia ROS production (and it also identified anti-oxidant compounds). Ofour 25 hits, we identified 10 pro-oxidants, 7 anti-oxidants, and 8eu-oxidants. Excluding the pro-oxidants, the remaining 15 compounds haveexcellent drug-like properties including a molecular weight between205-370, HBA<5, HBD<3, 1-3 rings, and a calculated Log S of >10-3. Thefinal screen is currently in progress and will identify compounds thatreverse hyperinsulinemia and hyperlipidemia-induced insulin resistancein cultured myotubes and adipocytes.

A flow chart of our screen is shown in Example 1, FIG. 1. The firstscreen identified compounds that increased energy expenditure and aselection of our hit compounds is shown in Example 1, FIG. 2. The secondscreen investigated the oxidant status of cells treated with 10 uM ofeach compound using fluorescence readout of the redox sensitive probeCM-DCFDA (Example 1, FIG. 3). Cellular ATP levels were measured after 70minutes at 10 uM dosage in order to rule out toxic compounds thatdiminish cellular ATP levels (Example 1, FIG. 4). Finally, since AMPKactivation has insulin-sensitizing effects (AMPK activation is linked tothe anti-diabetes effects of metformin, rosiglitazone, and berberine(3)) we assayed AMPK phosphorylation in Example 1, FIG. 4. These screensrevealed that compound BAM-15 was the most potent agonist of energyexpenditure, it increased oxygen consumption without ROS production, andit activated AMPK without depletion of ATP (Example 1, FIGS. 2-4). Thiscompound was used in a pilot study of five mice wherein it hadinsulin-sensitizing properties in mice fed a HFD.

1. To characterize novel energy expenditure agonists for the protectionof insulin resistance in vitro. To assess the ability of hit compoundsto reverse insulin resistance we will render L6 myotubes and 3T3-L1adipocytes insulin resistant with chronic hyperinsulinemia (4 treatmentsof 10 nM insulin over 24 h) or hyperlipidemia treatment (0.3 mMpalmitate for 24 h). These models of diabetes/insulin resistance arewell validated in our lab and have been described by us previously(4-5). Each hit compound will be co-treated with the insulin resistanceinsult over a time course (1-24 hours) and dose response (10 nM-10 μM).Insulin-stimulated signal transduction through the canonical insulinaction pathway will be monitored by Western blotting for phospho-IR,phospho-IRS1, phospho-S473/T308-Akt, and phospho-T162-AS160 as describedin (5). Additionally, GLUT4 translocation to the plasma membrane andglucose transport will be measured to assess insulin action. GLUT4trafficking will be measured as described in (5), and glucose uptakewill be measured using the 3H-2-deoxyglucose method described previously(6).

2: To test the insulin-sensitizing effects of novel energy expenditureagonists in high fat fed mice.

Pharmacokinetics and next generation energy expenditure libraries-Afterdeveloping the LCMS method to quantify compounds of interest, initial PKanalysis will use a one compartment IV bolus model to determineelimination rate constant, apparent volume of distribution, plasmahalf-life, clearance and area under the concentration vs. time curve.Plasma concentration measurements following dosing by oral gavage willbe used to calculate bioavailability. The most immediately useful ofthese parameters are half-life and oral availability, which will dictatefrequency of dosing (within 3 half lives) and route of administration,respectively. In the case of very short-lived compounds, we have theoption of delivery by mini-pump if the compound is sufficiently potent.We will engage in a structure-activity relationship study to improve thein vivo stability and/or oral availability of desirable compounds.

In vivo characterization of hit/lead compounds-Hit compound solubilitywill be determined experimentally by dilution in hydroxypropylβ-cyclodextrin (HPβCD) and other polyethylene glycol ormethylcellulose-based solvents. Dose escalation studies will beperformed to determine LD₅₀. One-half, one-tenth, and one-one hundredthof the LD₅₀ will be given to mice to assess drug efficacy by oxygenconsumption using CLAMS system. Upon validation of energy expenditure invivo we will select two dosages for each drug and commence in vivotesting for reversal of insulin resistance as described below. Hit andlead compounds will be chronically administered to high fat fed insulinresistant mice by i.p. injection, incorporation into food, or Azlet miniosmotic pumps (depending on oral bioavailability and pharmacokinetic(PK) properties). CLAMS respirometry will be used to measure energyexpenditure. Body weight, food intake, and glucose/insulin tolerancewill be measured every four weeks from the initiation of the high fatdiet.

We expect that approximately five compounds will be tested in vivo. Inthis scenario the first cohort for the in vivo study will include 12groups of five mice (60 mice). 7-week-old C57BL/6 mice will be orderedfrom Jackson labs. One group will remain on chow diet as a referencecontrol and 11 groups will be switched from a standard chow diet to anobesigenic and insulin resistance diet (60% fat, lard-based diet,Research Diets) for up to 12 weeks. After five weeks of high fatfeeding, mice will be assessed for baseline energy expenditure andinsulin sensitivity (by glucose tolerance test, insulin tolerance test,and fasting blood insulin/lipid measurements). We will then initiatepharmacologic intervention (e.g., via oral gavage or intraperitonealinjection) of the top five compounds at two dosages that have beenpredetermined to increase energy expenditure. The carrier control(solvent alone) will be administered to the remaining HFD group.Compounds will be given at two doses; a moderate-high dose less thanhalf of the LD50 that produces a>10% increase in energy expenditurewithout signs of physical distress, and a dose near the limit ofdetection of energy expenditure. Food intake and body weight will berecorded weekly. Insulin sensitivity will be reassessed every week byglucose tolerance testing. Mice will be maintained on compounds until aphenotype is apparent or up to 7 weeks (total of 12 weeks on HFD). Organweights will be recorded, including the liver and adipose depots, andlipid accumulation in liver will be determined by triglyceride assay andhistology with oil-red-o staining. Serum will be collected fordetermination of triglyceride, free fatty acid, cholesterol, insulin,and adiponectin. Successful compounds will show improvements in bloodlipid profiles, glucose tolerance, leanness, and insulin sensitivitywithout hypophagia or hyperinsulinemia.

Alternatives, contingency plans, and future studies-If hypophagia isobserved with the treatment of any compounds we will repeat thatcompound study with pair fed control mice to match the food intake ofthe test mice. Compounds that prevent insulin resistance in vivo will befurther investigated to determine the tissue-type that mediates thebeneficial effects. In brief, radiolabeled ¹⁴C-glucose and³H-2-deoxyglucose tracers will be administered during the GTT.Tissue-specific glucose transport will be monitored by extraction ofphospho-³H-2deoxyglucose, and glycogen synthesis will be determined by14C-glucose incorporation into glycogen) as described in (4, 7). Futurestudies will identify the molecular targets of the lead compounds. Wewill use SILAC-based quantitative proteomics for identification of thelead targets as described in (1). We have expertise in SILAC labeling aspublished previously (8).

A Seahorse Extracellular Flux Analyzer (XF24) is used for assessments ofoxygen consumption in intact cells. A SpectraMax M5 dual-monochromatormicroplate reader is used for the cell-based oxidative stress assays. ACLAMS indirect calorimeter is available. An ABI 4000 triple quadrupolemass spectrometer is used for the pharmacokinetic assessments.

BAM15 is a known chemical, but the uses described herein are new and theresults unexpected. It and the other useful compounds disclosed hereinor which are encompassed by the invention are an entirely new class ofmolecules that are acting as energy expenditure agonists. It wasarbitrarily named BAM15 in this laboratory. Its IUPAC name is(2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine,it is compound number ST056388 from Timtec. The library it came from isthe Timtec ApexScreen 5040. Experiments and procedures are being done tomodify and synthesize secondary amines.

Also disclosed herein are data that BAM15 and the other compounds of theinvention with similar activity are an entirely new class ofmitochondrial protonophore, others in this class are FCCP and2,4-dinitrophenol. BAM15 is superior to these molecules in some of ourassays both in terms of potency and toxicity. The methods of theinvention, however, include the use of compounds such as FCCP and2,4-dinitrophenol, alone or in combination.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

Example 1 Bibliography

1. Ong S E, Schenone M, Margolin A A, Li X, Do K, Doud M K, et al.Identifying the proteins to which small-molecule probes and drugs bindin cells. Proc Natl Acad Sci USA. 2009; 106(12):4617-22. PMCID: 2649954.

2. Tseng Y H, Cypess A M, Kahn C R. Cellular bioenergetics as a targetfor obesity therapy. Nat Rev Drug Discov. 2010; 9(6):465-82. PMCID:2880836.

3. Brunmair B, Staniek K, Gras F, Scharf N, Althaym A, Clara R, et al.Thiazolidinediones, like metformin, inhibit respiratory complex I: acommon mechanism contributing to their antidiabetic actions? Diabetes.2004; 53(4):1052-9.

4. Hoehn K L, Salmon A B, Hohnen-Behrens C, Turner N, Hoy A J, Maghzal GJ, et al. Insulin resistance is a cellular antioxidant defensemechanism. Proc Natl Acad Sci USA. 2009; 106(42):17787-92. PMCID:2764908.

5. Hoehn K L, Hohnen-Behrens C, Cederberg A, Wu L E, Turner N, Yuasa T,et al. IRS1-independent defects define major nodes of insulinresistance. Cell Metab. 2008; 7(5):421-33.

6. Yip M F, Ramm G, Larance M, Hoehn K L, Wagner M C, Guilhaus M, et al.CaMKII-mediated phosphorylation of the myosin motor Myolc is requiredfor insulin-stimulated GLUT4 translocation in adipocytes. Cell Metab.2008; 8(5):384-98.

7. Hoehn K L, Turner N, Swarbrick M M, Wilks D, Preston E, Phua Y, etal. Acute or chronic upregulation of mitochondrial fatty acid oxidationhas no net effect on whole-body energy expenditure or adiposity. CellMetab. 2010; 11(1):70-6.

8. Larance M, Rowland A F, Hoehn K L, Humphreys D T, Preiss T, GuilhausM, et al. Global phosphoproteomics identifies a major role for AKT and14-3-3 in regulating EDC3. Mol Cell Proteomics. 2010; 9(4):682-94.PMCID: 2860230.

Example 2

Mitochondrial Coupling

The primary pathway for energy production in aerobic cells involves theoxidation of nutrients in mitochondria via the tricarboxylic acid (TCA)cycle to produce CO₂ and high-energy electron carriers in the form ofNADH and FADH₂. NADH and FADH₂ donate electrons to the mitochondrialelectron transport chain (ETC) and activate a series of proton pumpsthat extrude protons from the mitochondrial matrix. Electrons reduce O₂at complex IV to form H₂O. This process creates a pH and electrochemicalgradient, also known as the proton motive force (pmf), across themitochondrial inner membrane (MIM). The major pathway for protonre-entry into the mitochondrial matrix is via ATP synthase resulting inATP production.

Mitochondrial Uncoupling

Protons that re-enter the mitochondrial matrix via pathways independentof ATP synthase ‘uncouple’ nutrient oxidation from ATP production.Uncoupling reduces the pmf and, therefore, increases the flow ofelectrons through the ETC as the mitochondria accelerate respiration tomaintain mitochondrial membrane potential. The reduced duration ofoccupancy of electrons at complexes I, II, and III decreases theinappropriate extraction of electrons by molecular oxygen at thesecomplexes and, thereby, decreases superoxide production. In contrast,high occupancy of electrons on electron carriers, caused byhyperpolarized mitochondria (for example, high ATP/low ADP ratio due tosedentary lifestyle or overnutrition), results in higher superoxideproduction⁷.

Uncoupling is a natural phenomenon that is mediated by a family ofuncoupling proteins (UCPs) found in the MIM. Uncoupling serves severalpurposes in cells. For example, UCP1 is highly expressed in brown fatwhere it becomes activated by cold stress to increase thermogenesis,whereas UCP2 is ubiquitous and has roles in the maintenance ofmitochondrial function and reduced oxidative stress⁸⁻⁹. For example, ourrecent work has demonstrated the requirement of UCP2 in phagocytosis andthe oxidation of corpse material¹⁰.

Chemical protonophores also transport protons across the MIM in theabsence of ATP generation and are very effective uncouplers. The twomost widely utilized chemical mitochondrial uncouplers are thehydrophobic weak acids DNP and carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP). Although DNP was prescribed tohumans and grossly overused, fatalities were rare. FCCP, which is morepotent than DNP, is rarely used in vivo.

One possible explanation for the toxicity observed with rosiglitazone,or the toxicity induced by aggressive maintenance of normoglycemia withinsulin treatment in the ACCORD and NICE-SUGAR trials may be due toincreased nutrient burden placed on peripheral tissues. For example,insulin sensitizers, such as rosiglitazone, and other insulin mimeticsstimulate glucose clearance into tissues such as skeletal and cardiacmuscle. This approach clears glucose from the blood by ‘pushing’ it intotissues. Although this ‘push’ approach has beneficial effects onlowering blood sugar levels its mechanism is in contrast to exercise,which depletes nutrient reserves and ‘pulls’ glucose from thecirculation. The present application discloses compositions and methodsfor improving glucose clearance by mimicking the ‘pull’ approach ofexercise while lessening the toxicity of non-selective protonophores.Advantages of using mitochondria-selective uncoupling to mediate thepull approach include decreased lipotoxicity, improved mitochondrialfunction, decreased ROS production, and decreased adiposity.

Example 2 Results

To identify new mitochondrial uncouplers with a broader therapeuticwindow, we developed a novel cell-based small molecule screeningapproach. The primary screen measured cellular oxygen consumption rate(OCR) in L6 myoblasts seeded in plates containing the oxygen-sensitivefluorophore 4,7-diphenyl-1,10-phenathroline ruthenium (II) chlorideembedded in silicone at the base of the well. This fluorophore isquenched by oxygen-thus fluorescence of each well increased as oxygen isconsumed by the cells. Each compound (library size was 5,040 compounds)was screened in duplicate at a concentration of ˜6 uM and 25 hits wereidentified that increased cellular oxygen consumption by at least 10%over baseline. Five of these hits were eliminated because they belongedto a family of known mitochondrial uncouplers. Twelve were eliminatedbecause they increased ROS production. The remaining 8 hits were testedfor cellular oxygen consumption across a dose range (10 nM to 10 μM). Asshown in Example 1, FIG. 2, we identified BAM15 as an exceptionalcompound with equal potency and a much greater therapeutic range thanFCCP.

A requirement of a bona-fide mitochondrial protonophore uncoupler is itsability to stimulate oxygen consumption when ATP synthase is inhibited.Treating L6 myoblasts with the ATP synthase inhibitor oligomycin haltsthe ETC by promoting a steep increase in pmf. This results in a largedecrease in oxygen consumption at complex IV (see Example 3).Mitochondrial protonophore uncouplers FCCP or BAM15 are able to increaseoxygen consumption in the presence of oligomycin because they increaseproton influx into the mitochondrial matrix to enable oxygen consumptionthrough complex IV. The 1 uM dose of BAM15 and FCCP was chosen becauseit represented a concentration where both compounds exhibited similarrespiration in cells treated without oligomycin (see Example 3). BAM15is far superior to FCCP at all higher doses (not shown). The ability touse BAM15 over a broad concentration range without toxicity is a keyadvance for the study of mitochondrial function because it avoids ‘dosefinding’ that must be achieved with FCCP to determine the maximalmitochondrial respiration rate and spare respiratory capacity.

Another requirement of a mitochondrial uncoupler is its ability tostimulate respiration in isolated mitochondria. To test this, weisolated mouse liver mitochondria (respiring on pyruvate/malate) andtreated them with BAM15 or FCCP. To determine that BAM15's mechanism ofaction was not due to electron donation to the ETC, we performed a‘complex coupling’ experiment in isolated mitochondria. As shown inExample 2, FIG. 1, the complex coupling experiment starts with isolatedmitochondria respiring on pyruvate and malate in the presence of FCCP orBAM15 (5 μM) at time 0. After 10 mins, 2 μM rotenone is added to inhibitETC complex I. Example 2, FIG. 1 shows that oxygen consumption ratesdropped in mitochondria treated with either FCCP or BAM15 indicatingthat they do not donate electrons to the ETC downstream of complex I.Succinate was then added at 20 min to stimulate respiration from complexII. Neither FCCP nor BAM15 affect the increase in respiration indicatingthat they do not affect complex II. At 25 min, the mitochondria weretreated with 4 μM antimycin A (AntA) to inhibit complex III and blocksuccinate-mediated respiration. These data demonstrate that neithercompound donates electrons from succinate to cytochrome c or complex IV.Finally, at 31 min the electron donor system of ascorbate/TMPD was addedto feed electrons to complex IV. In sum, these data indicate that BAM15increases respiration in isolated mitochondria via a mechanism that doesnot involve electron donation to the ETC.

The most significant problem with most currently available non-selectiveuncouplers, including FCCP and DNP, is their protonophore activity onnon-mitochondrial membranes. More specifically, inward protonconductance at the PM results in a substantial increase in energyexpenditure to restore resting membrane potential. The concentrations ofDNP or FCCP that are frequently used to induce maximal rates ofmitochondrial respiration (an index of mitochondrial function and sparerespiratory capacity) also promote PM depolarization resulting in adepletion of cellular ATP levels and the production of ROS leading tocaspase-dependent cell death. Intracellular acidification was determinedby loading L6 myotubes with the pH sensitive fluorophore SNARF-1 anddetermining ratiometric changes in fluorescence intensity against astandard curve. Mitochondrial uncouplers are expected to mildly increaseintracellular acidification due to the increased hydrolysis of ATP andinorganic phosphate production. Example 2, FIG. 2 illustrates theexpected mild acidification with BAM15 and shows that FCCP promotes2-fold more intracellular acidification, presumably due to its effectsat the PM.

To investigate whether the lack of protonophore activity at the PM andthe broad maximal efficacy range of BAM15 improved cell viability, wecultured L6 cells, NMuLi cells, and isolated neonatal rat ventricularcardiomyocytes in the presence of a dose range of FCCP or BAM15 for 48h. Cell number, morphology, and death were observed by phase contrastmicroscopy, MTT assay, and crystal violet staining. Example 2, FIGS.3A-B demonstrate the reduced cytotoxicity of BAM15, compared to FCCP, inall three groups of cells. Furthermore, FCCP resulted in a loss ofcellular ATP and activation of AMP-activated protein kinase (AMPK),whereas BAM15 treatment did not alter ATP levels and caused mild AMPKactivation (Example 2, FIG. 3C-D).

To determine whether BAM15 has biological activity in vivo, we initiateda pilot study wherein eight-week old C57BL/6 mice purchased from JacksonLaboratories were fed a 45% HFD for six weeks. Mice were tested forglucose tolerance and assigned to groups to normalize for similar bodymass and glucose tolerance. Mice were then treated daily by IP injectionof BAM15 (3 mg/kg ip) or an equal volume of carrier control (50%PEG400/5% DMSO) for seven days. At day 5, mice were subjected to a 2g/kg glucose tolerance test, and at day 8, mice were euthanized andtissue weights were analyzed. As shown in Example 2, FIG. 4, micetreated with 3 mg/kg BAM15 demonstrated improved glucose tolerance. Atnecropsy, the mice receiving 3 mg/kg BAM15 had reduced fat pad mass butnormal liver weight. These pilot data establish that BAM15 is a novelmitochondrial uncoupler and highlight the need for further developmentand in vivo testing.

Our in vivo phenotypic target is improved glucose tolerance andleanness. Mitochondrial uncoupling promotes leanness and insulinsensitivity. Therefore, the identification of safer uncouplers isrelevant to potential treatments for obesity and T2D.

BAM15 represents our prototype molecule for medicinal chemistry. We havesynthesized BAM15 and confirmed that its activity is identical toanalytically pure sample (data not shown).

Assays. Oxygen consumption rates in intact cells and isolatedmitochondria are determined using a Seahorse XF24 extracellular fluxanalyzer. This instrument provides a reproducible response and the dataobtained can be reliably used to validate and optimize BAM15derivatives. The first screen that will be performed with all newderivatives is a dose response of oxygen consumption in intact skeletalmuscle L6 myoblasts. All samples will be tested from 10 nM to 100 μM incomparison to both BAM15 and FCCP. New derivatives that have therapeuticwindow similar to or greater than BAM15 will be subjected to a ‘complexcoupling’ screen to validate that the compounds work on isolatedmitochondria and do not donate electrons directly to the electrontransport chain. New compounds that pass both of these filters will besubjected to the series of assays performed for BAM15 (see Example 2,FIG. 5) to test whether the new molecules behave as mitochondrialprotonophore uncouplers and have appropriate cell viability. Moleculesthat will be considered for in vivo testing will:

i. Not have protonophore activity at the PM or cause intracellularacidification. This will be tested by patch clamp analysis and pHiassays using SNARF-1 in the presence of NCEs. BAM15 (1 μM) and FCCP (1μM) will be used as controls for intracellular acidification.

ii. Not increase cellular ROS production. This will be tested by loadingcells with the ROS-sensitive dye CM-DCFDA and treating with the unknowncompounds. BAM15 (1 μM) will be used control for lack of ROS and thecomplex III inhibitor AntA (100 nM) will be used as a positive controlfor mitochondrial ROS production.

iii. Have greater cell viability than FCCP. Viability will be tested by48 hr treatment with doses ranging from 0.1-100 μM, as described inExample 2, FIG. 3. Cell viability and toxicity will be assessed by phasecontrast microscopy, LDH release, pro-caspase cleavage, MTT assay,and/or annexin V staining.

The preliminary studies described above demonstrate the intimatecollaboration of medicinal chemistry and pharmacology to illustrate theunprecedented activity of a small molecule mitochondria uncoupler BAM15.Our goal is to build upon these studies and develop compounds withimproved pharmacology so that they can be tested in mouse models ofdisease. Hence our strategy is straightforward: an iterative process ofchemical synthesis and in vitro and in vivo studies, includingpharmacokinetics (see for example, Example 4).

Through an iterative process of synthesis and biological testing, wewill create BAM15-like molecules that have a suitable half-life,potency, and bioavailability for testing in mouse models of obesity andinsulin resistance.

Improve Solubility Properties of BAM15.

To improve the solubility of BAM15 in aqueous buffer systems, we willintroduce solubilizing moieties while minimizing both structural andelectronic perturbations.

Define the Pharmacophore of BAM15

Because it is only now disclosed herein that BAM15 is a small moleculeprotonophore with unprecedented broad maximal efficacy range, SAR aroundthis molecule is limited. Hence, we will synthesize derivatives todevelop a pharmacophore that retains the desired activity. Experimentshave shown BAM15 to be more lipophilic than FCCP or 2,4-DNP.

Example 2 Bibliography

1. Del Prato S. Megatrials in type 2 diabetes. From excitement tofrustration? Diabetologia. 2009; 52(7):1219-26.

2. Stockton MTaA. Dinitrophenol in the treatment of obesity: Finalreport. JAMA. 1935; 5(105):332-7.

3. Colman E. Dinitrophenol and obesity: an early twentieth-centuryregulatory dilemma. Regul Toxicol Pharmacol. 2007; 48(2):115-7.

4. Caldeira da Silva C C, Cerqueira F M, Barbosa L F, Medeiros M H,Kowaltowski A J. Mild mitochondrial uncoupling in mice affects energymetabolism, redox balance and longevity. Aging Cell. 2008; 7(4):552-60.

5. Lou P H, Hansen B S, Olsen P H, Tullin S, Murphy M P, Brand M D.Mitochondrial uncouplers with an extraordinary dynamic range. Biochem J.2007; 407(1):129-40. PMCID: 2267406.

6. Heytler P G, Prichard W W. A new class of uncoupling agents-carbonylcyanide phenylhydrazones. Biochem Biophys Res Commun. 1962; 7:272-5.

7. Turrens J F. Superoxide production by the mitochondrial respiratorychain. Bioscience reports. 1997; 17(1):3-8.

8. Brand M D, Esteves T C. Physiological functions of the mitochondrialuncoupling proteins UCP2 and UCP3. Cell Metab. 2005; 2(2):85-93.

9. Mailloux R J, Harper M E. Mitochondrial proticity and ROS signaling:lessons from the uncoupling proteins. Trends Endocrinol Metab. 2012;23(9):451-8.

10. Park D, Han C Z, Elliott M R, Kinchen J M, Trampont P C, Das S,Collins S, Lysiak J J, Hoehn K L, Ravichandran K S. Continued clearanceof apoptotic cells critically depends on the phagocyte Ucp2 protein.Nature. 2011; 477(7363):220-4.

11. Wu Y N, Munhall A C, Johnson S W. Mitochondrial uncoupling agentsantagonize rotenone actions in rat substantia nigra dopamine neurons.Brain Res. 2011; 1395:86-93.

12. Pandya J D, Pauly J R, Sullivan P G. The optimal dosage and windowof opportunity to maintain mitochondrial homeostasis following traumaticbrain injury using the uncoupler FCCP. Exp Neurol. 2009; 218(2):381-9.

13. Korde A S, Pettigrew L C, Craddock S D, Maragos W F. Themitochondrial uncoupler 2,4-dinitrophenol attenuates tissue damage andimproves mitochondrial homeostasis following transient focal cerebralischemia. J Neurochem. 2005; 94(6): 1676-84.

14. Modriansky M, Gabrielova E. Uncouple my heart: the benefits ofinefficiency. J Bioenerg Biomembr. 2009; 41(2):133-6.

15. Brennan J P, Southworth R, Medina R A, Davidson S M, Duchen M R,Shattock M J. Mitochondrial uncoupling, with low concentration FCCP,induces ROS-dependent cardioprotection independent of KATP channelactivation. Cardiovasc Res. 2006; 72(2):313-21.

16. Murphy M P, Smith R A J. Targeting Antioxidants to Mitochondria byConjugation to Lipophilic Cations. Annual Review of Pharmacology andToxicology. 2007; 47(1):629-56.

17. Marrache S, Dhar S. Engineering of blended nanoparticle platform fordelivery of mitochondria-acting therapeutics. Proceedings of theNational Academy of Sciences. 2012; 109(40):16288-93.

18. Smith R A, Hartley R C, Murphy M P. Mitochondria-Targeted SmallMolecule Therapeutics and Probes. Antioxidants & Redox Signaling. 2011;15:3021-38.

19. Blaikie F H, Brown S E, Samuelsson L M, Brand M D, Smith R A, MurphyM P. Targeting dinitrophenol to mitochondria: limitations to thedevelopment of a self-limiting mitochondrial protonophore. BioscienceReports. 2006; 26(3):231-43.

20. Smith R A, Murphy M P. Animal and human studies with themitochondria-targeted antioxidant MitoQ. Annals of the New York Academyof Sciences. 2010; 1201:96-103.

21. Rodriguez-Cuenca S, Cochemé H M, Logan A, Abakumova I, Prime T A,Rose C, Vidal-Puig A, Smith A C, Rubinsztein D C, Fearnley I M, Jones BA, Pope S, Heales S J R, Lam B Y H, Neogi S G, McFarlane I, James A M,Smith R A J, Murphy M P. Consequences of long-term oral administrationof the mitochondria-targeted antioxidant MitoQ to wild-type mice. FreeRadical Biology and Medicine. 2010; 48(1):161-72.

22. Horton K L, Stewart K M, Fonseca S B, Guo Q, Kelley S O.Mitochondria-Penetrating Peptides. Chemistry and Biology. 2008;15(4):375-82.

23. Zhao K, Zhao G-M, Wu D, Soong Y, Birk A V, Schiller P W, Szeto H H.Cell-permeable Peptide Antioxidants Targeted to Inner MitochondrialMembrane inhibit Mitochondrial Swelling, Oxidative Cell Death, andReperfusion Injury. Journal of Biological Chemistry. 2004;279(33):34682-90.

24. Bryson D I, Zhang W, Ray W K, Santos W L. Screening of a BranchedPeptide Library with HIV-1 TAR RNA. Mol BioSyst. 2009; 5:1070-3.

25. Bryson D I, Zhang W, McLendon P M, Reineke T M, Santos W L. Towardtargeting RNA structure: branched peptides as cell-permeable ligands toTAR RNA. ACS Chemical Biology. 2012; 7(1):210-7. PMCID: 3262918.

26. Hoehn K L, Hohnen-Behrens C, Cederberg A, Wu L E, Turner N, Yuasa T,Ebina Y, James D E. IRS1-independent defects define major nodes ofinsulin resistance. Cell Metab. 2008; 7(5):421-33.

27. Hoehn K L, Salmon A B, Hohnen-Behrens C, Turner N, Hoy A J, MaghzalG J, Stocker R, Van Remmen H, Kraegen E W, Cooney G J, Richardson A R,James D E. Insulin resistance is a cellular antioxidant defensemechanism. Proc Natl Acad Sci USA. 2009; 106(42):17787-92. PMCID:2764908.

28. Hoehn K L, Turner N, Swarbrick M M, Wilks D, Preston E, Phua Y,Joshi H, Furler S M, Larance M, Hegarty B D, Leslie S J, Pickford R, HoyA J, Kraegen E W, James D E, Cooney G J. Acute or chronic upregulationof mitochondrial fatty acid oxidation has no net effect on whole-bodyenergy expenditure or adiposity. Cell Metab. 2010; 11(1):70-6.

29. MacArthur D G, Seto J T, Chan S, Quinlan K G, Raftery J M, Turner N,Nicholson M D, Kee A J, Hardeman E C, Gunning P W, Cooney G J, Head S I,Yang N, North K N. An Actn3 knockout mouse provides mechanistic insightsinto the association between alpha-actinin-3 deficiency and humanathletic performance. Hum Mol Genet. 2008; 17(8): 1076-86.

30. Patel S A, Hoehn K L, Lawrence R T, Sawbridge L, Talbot N A, TomsigJ L, Turner N, Cooney G J, Whitehead J P, Kraegen E W, Cleasby M E.Overexpression of the Adiponectin Receptor AdipoR1 in Rat SkeletalMuscle Amplifies Local Insulin Sensitivity. Endocrinology. 2012.

31. Bonnard C, Durand A, Peyrol S, Chanseaume E, Chauvin M A, Morio B,Vidal H, Rieusset J. Mitochondrial dysfunction results from oxidativestress in the skeletal muscle of diet-induced insulin-resistant mice. JClin Invest. 2008; 118(2):789-800.

32. Sauerbeck A, Pandya J, Singh I, Bittman K, Readnower R, Bing G,Sullivan P. Analysis of regional brain mitochondrial bioenergetics andsusceptibility to mitochondrial inhibition utilizing a microplate basedsystem. J Neurosci Methods. 2011; 198(1):36-43. PMCID: 3535268.

33. Djouadi F, Riveau B, Merlet-Benichou C, Bastin J. Tissue-specificregulation of medium-chain acyl-CoA dehydrogenase gene by thyroidhormones in the developing rat. Biochem J. 1997; 324 (Pt 1):289-94.PMCID: 1218429.

34. Bruce C R, Thrush A B, Mertz V A, Bezaire V, Chabowski A,Heigenhauser G J, Dyck D J. Endurance training in obese humans improvesglucose tolerance and mitochondrial fatty acid oxidation and altersmuscle lipid content. Am J Physiol Endocrinol Metab. 2006;291(1):E99-E107.

35. Turner N, Bruce C R, Beale S M, Hoehn K L, So T, Rolph M S, Cooney GJ. Excess lipid availability increases mitochondrial fatty acidoxidative capacity in muscle: evidence against a role for reduced fattyacid oxidation in lipid-induced insulin resistance in rodents. Diabetes.2007; 56(8):2085-92.

36. Kim J Y, Hickner R C, Cortright R L, Dohm G L, Houmard J A. Lipidoxidation is reduced in obese human skeletal muscle. Am J PhysiolEndocrinol Metab. 2000; 279(5):E1039-44.

37. Lin L, Saha PK, Ma X, Henshaw I O, Shao L, Chang B H, Buras E D,Tong Q, Chan L, McGuinness O P, Sun Y. Ablation of ghrelin receptorreduces adiposity and improves insulin sensitivity during aging byregulating fat metabolism in white and brown adipose tissues. AgingCell. 2011; 10(6):996-1010. PMCID: 3215833.

Example 3

Oxidative phosphorylation in mitochondria is intrinsic to energyproduction in aerobic eukaryotic cells. This process, which isdiagrammed in Example 3, FIG. 1A, involves the coupling of nutrientoxidation to ATP production through a proton cycle across themitochondrial inner membrane. Any pathway that enables proton re-entryinto the matrix independent of ATP synthase ‘uncouples’ nutrientoxidation from ATP production. Mild uncoupling has antioxidant effectsby lessening the proton motive force and shortening the occupancy timeof single electrons at electron carriers within the electron transportchain. In contrast, a high proton motive force leads to a more reducedstate of the electron transport chain and increases the rate ofmitochondrial superoxide production (1-3). Genetic and pharmacologicuncoupling have beneficial effects on disorders that are linked tomitochondrial oxidative stress (i.e. ischemic-reperfusion injury (4-7),Parkinson's disease (8), insulin resistance (9-10), aging (11), andheart failure (12)) and disorders that stand to benefit from increasedenergy expenditure such as obesity (13).

The two most widely utilized chemical uncouplers, 2,4-dinitrophenol(DNP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),were discovered more than 50 years ago but remain the reagents of choicefor mitochondrial bioenergetics studies. FCCP is more potent than DNPand is preferred for the study of mitochondrial function, whereas DNP isless potent and has more utility in vivo. One of the most significantlimitations of DNP and FCCP is thought to be their proton transporter(protonophore) activity at the plasma membrane (14-16). Protonophoreactivity at the plasma membrane induces depolarization and leads to arange of off-target effects including the opening of voltage-sensitiveion channels (17). In some cells, more than 50% of cellular energyexpenditure is used to maintain cellular ion gradients (18), thereforethe combination of chronic plasma membrane depolarization with reducedefficiency of mitochondrial ATP production leads to increasedcytotoxicity at high concentrations.

Example 3 Results

To identify new mitochondrial uncouplers with low toxicity, we developeda cell-based small molecule screen. Our primary screen identifiedmolecules that increased cellular oxygen (O₂) consumption, an indicatorof increased oxidative phosphorylation. In this assay, L6 myoblast cellswere seeded into a 96-well plate containing an O₂-sensitive fluorophoreembedded in silicone at the base of the well (Example 3, FIG. S1). Eachcompound was screened at a concentration of 5 μg/mL and FCCP was used asa positive control (Example 3, FIG. S1B). Positive hits were subjectedto a secondary screen to identify, and eliminate, those that increasedcellular oxygen consumption via the production of reactive oxygenspecies (ROS) (Example 3, FIG. S2). Hit compounds that did not increaseROS production and were structurally unrelated to known uncouplers werefurther tested across a concentration range from 10 nM to 10 μM todetermine their effective dosing index. This algorithm identified BAM15as an oxygen consumption agonist (Example 3, FIG. S1C) that reducedcellular ROS (Example 3, FIG. S2) and had a broad dynamic range inmyoblasts, primary neonatal rat ventricular cardiomyocytes, and normalmurine liver cells (Example 3, FIGS. 1C and S3). The high rates ofrespiration induced by BAM15 were accompanied by a proportional increasein the rate of extracellular acidification; which is a correlativemeasure of both glycolysis and the condensation of CO₂ with H₂O to formHCO3⁻ and H⁺ from nutrient oxidation (Example 3, FIG. S4).

BAM15 was next subjected to a series of biochemical assays to determinewhether it possessed properties required of a mitochondrial protonophoreuncoupler. These assays tested the ability of BAM15 to: 1) stimulate OCRwhen ATP synthase is inhibited; 2) depolarize the mitochondrial innermembrane; 3) stimulate respiration in isolated mitochondria; and 4)increase OCR without donating electrons to the electron transport chain.As shown in FIGS. 1D-F, BAM15 met all of these criteria.

First, BAM15 stimulated mitochondrial respiration in the presence of theATP synthase inhibitor oligomycin in L6 myoblasts (Example 3, FIG. 1D).In this experiment, BAM15 and FCCP were used at an equipotentconcentration of 1 μM so that a direct comparison between these twouncouplers could be made. However, BAM15 achieved higher rates ofrespiration than FCCP in both L6 and NMuLi cells when the uncouplerswere administered at 2 μM or 10 μM (Example 3, FIG. S5). Second, BAM15treatment of L6 myoblasts depolarized mitochondria, as demonstrated by aleftward shift in fluorescence of the cationic mitochondrial membranepotential dye TMRM (Example 3, FIG. 1E).

In light of the broad dynamic range we observed for BAM15, we tested theeffects of BAM15 and FCCP on plasma membrane electrophysiology of L6cells using whole cell patch clamp recordings. Under voltage clamp, FCCPinduced an inward current at a holding potential of −70 mV that wasfully recoverable upon washout, and repeatable upon multipleapplications. Moreover, the FCCP-induced current was dose-dependent andassociated with an increase in conductance (Example 3, FIGS. 2A-F). Incontrast, BAM15 elicited no appreciable change in current in the samecells at either concentration and across a broad voltage range. OnlyFCCP caused reversible and repeatable plasma membrane depolarizationunder current clamp at the resting membrane potential (Example 3, FIGS.2G-H), and with current injection to produce a membrane potential at −70mV (data not shown). The differential effects of BAM15 and FCCP onplasma membrane properties were independent of the order of uncouplerapplication (data not shown). These data indicate that BAM15 does notshare the adverse plasma membrane effects that are thought to restrictthe use of FCCP.

Since BAM15 is devoid of plasma membrane protonophore activity, weexamined relative cell viability following BAM15 treatment. Cellviability was determined following 48 hours exposure to increasing dosesof BAM15 or FCCP (up to 50 μM). BAM15 was 2- to 4-fold less cytotoxicthan FCCP in cultured myoblasts, hepatocytes and cardiomyocytes (Example3, FIG. 3). Representative examples of crystal violet staining and phasecontrast microscopy images demonstrate the differences in cell numberand morphology in BAM15-treated wells, as compared to equimolarconcentrations of FCCP (Example 3, FIGS. 3A-B).

One of the established biological uses of mitochondrial uncouplers isprotection from ischemic reperfusion injury (7, 21). Uncoupling reducesreperfusion-induced mitochondrial oxidative stress and mitochondrialfragmentation (4, 22). For example, ischemic pre-conditioning requiresupregulation of uncoupling protein 2 (UCP2) to prevent ischemicreperfusion injury (5, 7). Given the potent and selective uncouplingactivity of BAM15, we tested its therapeutic potential in vivo using amouse model of renal ischemic reperfusion injury. In this model, BAM15was administered as a single intraperitoneal bolus at 1 mg/kg or 5 mg/kgone hour prior to 26 minutes of bilateral renal ischemia and 48 hours ofreperfusion. Compared to vehicle, mice treated with BAM15 were protectedfrom kidney damage as determined by a dose-dependent decrease in plasmacreatinine levels at 24 and 48 h post-ischemia (Example 3, FIG. 4A).Histological analysis of H&E stained kidney outer medulla demonstratedthat BAM15 treatment markedly reduced tubular necrosis, depletion ofbrush border villi, and obstruction of proximal tubules (Example 3, FIG.4B-C). Furthermore, mice pre-treated with BAM15 had a dose-dependentdecrease in leukocyte infiltration compared to vehicle controls (Example3, FIG. S6).

In summary, we report the identification of BAM15 as a new chemotype ofmitochondrial protonophore uncoupler. BAM15 is highly potent anddemonstrates a greater maximally effective dynamic range than thegold-standard uncoupler FCCP. Unlike FCCP, BAM15 depolarizesmitochondria without affecting plasma membrane potential. Thesequalities allow BAM15 to sustain maximal rates of mitochondrialrespiration with low cytotoxicity, and enable the study of mitochondrialfunction in intact cells without interference from off-target effects atthe plasma membrane. Furthermore, compared with FCCP, BAM15 stimulatesgreater maximal cellular respiration in most cell lines tested (Example3, FIG. S3), suggesting that FCCP may underestimate maximalmitochondrial respiration due to toxicity. Finally, the ability of BAM15to protect from renal ischemic reperfusion injury demonstratespre-clinical efficacy and provides renewed optimism that protonophoresmay again be useful for medical intervention in the myriad disorderslinked to mitochondrial dysfunction.

Example 3 Bibliography

1. S. S. Korshunov, V. P. Skulachev, A. A. Starkov, FEBS Lett 416, 15(Oct. 13, 1997).

2. J. F. Turrens, Biosci Rep 17, 3 (February, 1997).

3. C. L. Quinlan et al., J Biol Chem 287, 27255 (Aug. 3, 2012).

4. M. N. Sack, Cardiovasc Res 72, 210 (Nov. 1, 2006).

5. C. J. McLeod, A. Aziz, R. F. Hoyt, Jr., J. P. McCoy, Jr., M. N. Sack,J Biol Chem 280, 33470 (Sep. 30, 2005).

6. A. S. Korde, L. C. Pettigrew, S. D. Craddock, W. F. Maragos, JNeurochem 94, 1676 (September, 2005).

7. M. Modriansky, E. Gabrielova, J Bioenerg Biomembr 41, 133 (April,2009).

8. Y. N. Wu, A. C. Munhall, S. W. Johnson, Brain Res 1395, 86 (Jun. 13,2011).

9. E. J. Anderson et al., J Clin Invest, (Feb. 2, 2009).

10. K. L. Hoehn et al., Proc Natl Acad Sci USA 106, 17787 (Oct. 20,2009).

11. C. C. Caldeira da Silva, F. M. Cerqueira, L. F. Barbosa, M. H.Medeiros, A. J. Kowaltowski, Aging Cell 7, 552 (August, 2008).

12. J. P. Brennan et al., Cardiovasc Res 72, 313 (Nov. 1, 2006).

13. Y. H. Tseng, A. M. Cypess, C. R. Kahn, Nat Rev Drug Discov 9, 465(June, 2010).

14. K. S. Park et al., Pflugers Arch 443, 344 (January, 2002).

15. S. K. Juthberg, T. Brismar, Cell Mol Neurobiol 17, 367 (August,1997).

16. T. Brismar, V. P. Collins, J Physiol 460, 365 (January, 1993).

17. K. J. Buckler, R. D. Vaughan-Jones, J Physiol 513 (Pt 3), 819 (Dec.15, 1998).

18. C. Howarth, P. Gleeson, D. Attwell, J Cereb Blood Flow Metab 32,1222 (July, 2012).

19. G. W. Rogers et al., PLoS One 6, e21746 (2011).

20. P. H. Lou et al., Biochem J 407, 129 (Oct. 1, 2007).

21. E. Y. Plotnikov et al., Biochemistry (Mosc) 77, 1029 (September,2012).

22. M. Zhan, C. Brooks, F. Liu, L. Sun, Z. Dong, Kidney Int 83, 568(April, 2013).

23. A. S. Divakaruni et al., Proc Natl Acad Sci USA, (Mar. 19, 2013).

24. L. Li et al., J Clin Invest 122, 3931 (Nov. 1, 2012).

25. A. Bajwa et al., J Am Soc Nephrol 21, 955 (June, 2010). 26. L. Li etal., J Immunol 178, 5899 (May 1, 2007).

Example 4

Experiments were performed to measure the amount of BAM15 in the bloodfollowing oral administration. Mice were provided BAM15 orally at 5mg/kg and then drug plasma levels were measured over time. Thepharmacokinetic analysis is demonstrated in Example 4, FIG. 1.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

What is claimed is:
 1. A method for regulating glucose homeostasis ortreating type II diabetes, the method comprising administering to asubject in need thereof a pharmaceutical composition comprising apharmaceutically acceptable carrier, optionally at least one additionaltherapeutic agent, and an effective amount of at least one compoundhaving a structure of Formula I or Formula II:

wherein R₁-R₁₀ are all independently optional, and are eachindependently selected from the group consisting of H, halogen, hydroxy,acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, and amino acid, whereineach group can be optionally substituted, or a pharmaceuticallyacceptable salt thereof, or

wherein R₁-R₂ are independently optional, and are each independentlyselected from the group consisting of H, halogen, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, and amino acid, wherein each group can beoptionally substituted, or a pharmaceutically acceptable salt thereof.2. The method of claim 1, wherein said compound is selected from thegroup consisting of:


3. The method of claim 1, wherein said compound is administered at adosage ranging from about 0.1 mg/kg to about 50 mg/kg body weight. 4.The method of claim 3, wherein said compound is administered at a dosageranging from about 0.5 mg/kg to about 25 mg/kg body weight.
 5. Themethod of claim 4, wherein said compound is administered at a dosageranging from about 1.0 mg/kg to about 5.0 mg/kg body weight.
 6. Themethod of claim 3, wherein said compound is administered as a unit doseranging from about 10 mg to about 500 mg.
 7. The method of claim 1,wherein said compound is administered more than once.
 8. The method ofclaim 1, wherein said compound is BAM15:


9. The method of claim 1, wherein the method is a method for regulatingglucose homeostasis and the subject is a human.
 10. The method of claim1, wherein the disease, disorder, or condition is type II diabetes andthe subject is a human.